Genetic loci associated with mechanical stalk strength in maize

ABSTRACT

The invention relates to methods and compositions for identifying and for selecting maize plants with mechanical stalk strength characteristics. The methods use molecular markers to identify and select plants with increased mechanical stalk strength or to identify and counter-select plants with decreased mechanical stalk strength. Maize plants generated by the methods of the invention are also a feature of the invention.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of U.S. application Ser. No.12/502,365, filed Jul. 14, 2009, which claims the benefit of U.S.Provisional Application No. 61/080,783, filed Jul. 15, 2008, which isincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods useful inenhancing mechanical stalk strength in maize plants.

BACKGROUND OF THE INVENTION

In maize, stalk lodging, or stalk breakage, accounts for significantannual yield losses in the United States. During a maize plant'svegetative growth phase, rapid growth weakens cell walls, making stalktissue brittle and increasing the propensity for stalks to snap whenexposed to strong, sudden winds and/or other weather conditions. Thistype of stalk lodging, called green snap or brittle snap, typicallyoccurs at the V5 to V8 stage, when the growing point of a maize plant isemerging from the soil line, or at the V12 to R1 stage, about two weeksprior to tasseling and until just after silking. Another type of stalklodging, late season stalk lodging occurs near harvest when the stalkcannot support the weight of the ear.

Factors that weaken the stalk during late season include insect attack,such as the European corn borer tunneling into stalk and ear shanks, andinfection by pathogens such as Colletotrichum graminicola, the causativeagent in Anthracnose stalk rot. Adverse fall weather conditions alsocontribute to late season stalk lodging.

The mechanical strength of the maize stalk plays a major role in aplant's resistance to all types of stalk lodging, and therefore, is ofgreat value to the farmer. Enhancing overall mechanical stalk strengthin maize will make stalks stronger during both vegetative developmentand late season, thereby reducing yield and grain quality losses.Moreover, maize plants with increased mechanical stalk strength canremain in the field for longer periods of time, allowing farmers todelay harvest, if necessary.

Selection through the use of molecular markers associated withmechanical stalk strength has the advantage of permitting at least someselection based solely on the genetic composition of the progeny, andselections can be made very early on in the plant life cycle, even asearly as the seed stage. The increased rate of selection that can beobtained through the use of molecular markers associated with mechanicalstalk strength means that plant breeding for increased mechanical stalkstrength can occur more rapidly.

It is desirable to provide compositions and methods for identifying andselecting maize plants that display overall increased mechanical stalkstrength.

SUMMARY

Compositions and methods for selecting maize plants with mechanicalstalk strength characteristics are provided herein, includingcompositions and methods for identifying and selecting maize plants withincreased mechanical stalk strength and compositions and methods foridentifying and counter-selecting maize plants that have decreasedmechanical stalk strength.

In one embodiment, methods of selecting a maize plant or germplasm withmechanical stalk strength characteristics are provided. In thesemethods, DNA is obtained, and the presence of at least one marker alleleis detected. The marker allele can include any marker allele that islinked to and associated with any of the following marker alleles: a “G”at PHM3468.1, a “T” at PHM3468.4, a “G” at PHM3468.18, a “T” atPHM12521.12, a “C” at PHM10840.105, an “A” at PHM10840.118, a “C” atPHM10840.130, a “C” at PHM16736.6, an “A” at PHM16736.14, a “C” atPHM14053.7, a “C” at PHM14053.8, a “C” at PHM14053.14, a “T” atPHM405.35, a “C” at PHM12025.26, a “T” at PHM18693-9-U, a “G” atPHM10786-11-U, a “C” at PHM10786-5-U, a “T” at PHM10786-6-U, a “G” atPHM8057-801-U, a “C” at PHM201-16-U, a “C” or a “G” at PHM201-17-U, a“T” or a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” atPHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an“A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” at C00386-397-U, a“C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” atPHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T”at PHM11186-16-V. A maize plant or germplasm that has the marker allelelinked to and associated with any of the marker alleles listed above isthen selected.

In other embodiments, the marker allele can be linked to any of thefollowing marker alleles: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G”at PHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” atPHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” atPHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” atPHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” atPHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” atPHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” or a“G” at PHM201-17-U, a “T” or a “G” at PHM4861-20-U, an “A” atPHM4861-21-U, a “G” at PHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a“T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a“C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T”at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” atPHM12025-48, and a “T” at PHM 11186-16-V by 30 cM, 25, 20, 15, 10, 9, 8,7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 cM.

In other embodiments, the marker allele can be any of the followingmarker alleles: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G” atPHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” atPHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” atPHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” atPHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” atPHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” atPHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” or a“G” at PHM201-17-U, a “T” or a “G” at PHM4861-20-U, an “A” atPHM4861-21-U, a “G” at PHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a“T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a“C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T”at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” atPHM12025-48, and a “T” at PHM11186-16-V.

In another embodiment, methods of selecting a maize plant or germplasmwith mechanical stalk strength characteristics are provided. In thesemethods, DNA is obtained, and the absence of at least one marker alleleis detected. The marker allele can include any marker allele that islinked to and associated with any of the following marker alleles: a “T”at PHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” atPHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” atPHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” atPHM14080-16-V, a “C” at PHM15089-10-U, and a “G” at PHM9364-6-U. A maizeplant or germplasm that does not have the marker allele linked to andassociated with any of the marker alleles listed above is then selected.

In other embodiments, the marker allele can be linked to any of thefollowing marker alleles: a “T” at PHM2130.24, an “A” at PHM2130.29, a“C” at PHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” atPHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” atPHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G”at PHM9364-6-U, by 30 cM, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1,0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 cM.

In other embodiments, the marker allele can be any of the followingmarker alleles: a “T” at PHM2130.24, an “A” at PHM2130.29, a “C” atPHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” atPHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” atPHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G”at PHM9364-6-U.

In another embodiment, methods for identifying maize plants withincreased or decreased mechanical stalk strength by detecting at leastone marker allele associated with increased or decreased mechanicalstalk strength in the germplasm of a maize plant are provided. Themarker locus can be selected from any of the marker loci provided inTable 3 or Table 7, as well as any other marker that is linked to thesemarkers. The marker locus can be found within any of the followingchromosomal intervals on linkage group 5, comprising and flanked by:

-   -   (i) PHM654 and PHM6727;    -   (ii) PHM12632 and PHM3323;    -   (iii) PHM201 and PHM3323; and    -   (iv) PHM201 and PHM3468.        More than one marker locus can be selected in the same plant        with no limitation as to which markers are selected in        combination. The markers used in combinations can be any of the        markers in Table 3 or Table 7, any other marker linked to the        markers in Table 3 or Table 7 (e.g., the linked markers        determined from theMaizeGDB resource), or any marker within the        intervals described herein.

In another embodiment, methods of selecting maize plants with increasedmechanical stalk strength are provided. In one aspect, a first maizeplant is obtained that has at least one allele of a marker locus whereinthe allele is associated with increased mechanical stalk strength. Themarker locus can be found within any of the following chromosomalintervals on linkage group 5, comprising and flanked by:

-   -   (i) PHM654 and PHM6727;    -   (ii) PHM12632 and PHM3323;    -   (iii) PHM201 and PHM3323; or    -   (iv) PHM201 and PHM3468.        The first maize plant is crossed to a second maize plant, and        the progeny plants resulting from the cross are evaluated for        the allele of the first maize plant. Progeny plants that possess        the allele from the first maize plant can then be selected as        having increased mechanical stalk strength.

In another embodiment, methods for not selecting plants with decreasedmechanical stalk strength are provided. In one aspect, a first maizeplant is obtained that has at least one allele of a marker locus whereinthe allele is associated with decreased mechanical stalk strength. Themarker locus can be found within any of the following chromosomalintervals on linkage group 5, comprising and flanked by:

-   -   (i) PHM654 and PHM6727;    -   (ii) PHM12632 and PHM3323;    -   (iii) PHM201 and PHM3323; and    -   (iv) PHM201 and PHM3468.        The first maize plant is crossed to a second maize plant, and        the progeny plants resulting from the cross are evaluated for        the allele of the first maize plant. Progeny plants that possess        the allele from the first maize plant can be identified as        having decreased mechanical stalk strength and can be removed        from a breeding program or planting.

In another embodiment, methods for selecting a maize plant withmechanical stalk strength characteristics are provided in which at leastone marker locus is assayed within the maize plant. The marker locus canbe located:

a. on chromosome 1, within the interval comprising and flanked byPHM7844 and PHM8029;

b. on chromosome 1, within the interval comprising and flanked byPHM7844 and PHM574;

c. on chromosome 1, within the interval comprising and flanked byPHM11754 and PHM1481;

d. on chromosome 1, within the interval comprising and flanked byPHM6427 and PHM1481;

e. on chromosome 1, within the interval comprising and flanked byPHM11125 and PHM13958;

f. on chromosome 1, within the interval comprising and flanked byPHM10468 and PHM13958;

g. on chromosome 9, within the interval comprising and flanked byPHM4578 and PHM11186; or

h. on chromosome 9, within the interval comprising and flanked byPHM14053 and PHM16736; and is associated with mechanical stalk strength.The maize plant is then selected if it possesses a favorable allele atthe marker locus.

In another embodiment, methods for selecting a maize plant withmechanical stalk strength characteristics are provided in which at leastone marker locus is assayed within the maize plant. The marker locus canbe located:

a. on chromosome 1, within the interval comprising and flanked byPHM7844 and PHM8029;

b. on chromosome 1, within the interval comprising and flanked byPHM7844 and PHM574;

c. on chromosome 1, within the interval comprising and flanked byPHM11754 and PHM1481;

d. on chromosome 1, within the interval comprising and flanked byPHM6427 and PHM1481;

e. on chromosome 1, within the interval comprising and flanked byPHM11125 and PHM13958;

f. on chromosome 1, within the interval comprising and flanked byPHM10468 and PHM13958;

g. on chromosome 9, within the interval comprising and flanked byPHM4578 and PHM11186; or

h. on chromosome 9, within the interval comprising and flanked byPHM14053 and PHM16736; and is associated with mechanical stalk strength.The maize plant is then selected if it does not possess an unfavorableallele at the marker locus.

Maize plants identified or selected by any of the methods describedherein are also included.

BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTINGS

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

FIGS. 1A-I show the physical map arrangement of sequenced BACs (obtainedfrom the Maize Genome Browser, which is publicly available on theinternet; http://www.maizesequence.org) that assemble to the chromosome5 region defined by and including BACs c0216105 and c0117h02. Thepositions of the markers listed in Table 7 are also indicated.

FIG. 2A shows a structured association analysis, wherein chromosome 5markers were tested for significance of association with mechanicalstalk strength. Mechanical stalk strength values were obtained using anInstron™ machine and a three-point bend test, on a set of 189 lines.Stalk strength values were acquired from plants in late season. X axis:Distance expressed in cM on Chr. 5. Y axis: probability value.

FIG. 2B shows an association analysis of an NSS subpopulation, whereinchromosome 5 markers were tested for significance of association withmechanical stalk strength. The NSS subpopulation consisted of 60 lines,which varied in maturity from a CRM (comparative relative maturity) of105 to a CRM of 110. Mechanical stalk strength values were obtainedusing an Instron™ machine and a three-point bend test and were acquiredfrom plants in late season. X axis: Distance expressed in cM on Chr. 5.Y axis: probability value.

FIGS. 2C-E show the clusters of markers on chromosome 5 thatco-segregate with mechanical stalk strength in the NSS subpopulation atthe following p-levels: C) a p-level of ≦0.01 (white data pointsrepresent the region defined by and including PHM654 and PHM6727), D) ap-level of ≦0.001 (white data points represent the region defined by andincluding PHM12632 and PHM3323, and E) a p-level of ≦0.0001 (white datapoints represent the region defined by and including PHM201 andPHM3323). Black dots represent associated markers that do not fallwithin each respective cluster.

FIG. 3 shows associations between chromosome 9 marker loci andmechanical stalk strength in the NSS subpopulation. The NSSsubpopulation consisted of 60 lines, which varied in maturity from a CRM(comparative relative maturity) of 105 to a CRM of 110. Mechanical stalkstrength values were obtained using an Instron™ machine and athree-point bend test and were acquired from plants in late season. Xaxis: Distance expressed in cM on Chr. 9. Y axis: probability value.

FIG. 4 shows associations between chromosome 1 marker loci andmechanical stalk strength in the NSS subpopulation. The NSSsubpopulation consisted of 60 lines, which varied in maturity from a CRM(comparative relative maturity) of 105 to a CRM of 110. Mechanical stalkstrength values were obtained using an Instron™ machine and athree-point bend test and were acquired from plants in late season. Xaxis: Distance expressed in cM on Chr. 1. Y axis: probability value.

FIG. 5 shows the PHM marker alleles for A) PHM201, B) PHM5421, and C)PHM3468. The positions of the polymorphisms relative to the referencesequence are represented in the numbers at the top of each table.

FIG. 6 depicts a plot of the average mechanical stalk strength valuesfor: A) haplotypes constituting marker alleles at PHM201 and at PHM5421and B) PHM3468 marker alleles.

FIG. 7 shows the composite interval mapping results for data set 1(portable Instron™ data) using the B73×Mo17 (IBM) syn4 population. Apeak of significance was identified on chromosome 5. Marker positions onthe x-axis correspond to the modified IBM2 genetic map. The y-axisrepresents the LOD score.

FIG. 8 shows the composite interval mapping results for data set 2(device and method described in patent application US2007/0125155(published Jun. 6, 2007)) using the B73×Mo17 (IBM) syn4 population. Apeak of significance was identified on chromosome 5. Marker positions onthe x-axis correspond to the modified IBM2 genetic map. The y-axisrepresents the LOD score.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

Table 1 lists the sequences described herein that are associated withthe PHM markers, along with the corresponding identifiers (SEQ ID NO:)as used in the attached Sequence Listing.

TABLE 1 PHM Marker Sequences Reference sequence Marker (SEQ ID ForwardReverse Locus NO:) (SEQ ID NO:) PHM654 1 Internal 40 41 External 42 43PHM111 2 Internal 44 45 External 46 47 PHM10100 3 Internal 48 49External 50 51 PHM7357 4 Internal 52 53 External 54 55 PHM5349 5Internal 56 57 External 58 59 PHM4167 6 Internal 60 61 External 62 63PHM14947 7 Internal 64 65 External 66 67 PHM5266 8 Internal 68 69External 70 71 PHM12632 9 Internal 72 73 External 74 75 PHM201 10Internal 76 77 External 78 79 PHM4861 11 Internal 80 81 External 82 83PHM5421 12 Internal 84 85 External 86 87 PHM4115 13 Internal 88 89External 90 91 PHM12521 14 Internal 92 93 External 94 95 PHM3468 15Internal 96 97 External 98 99 PHM10840 16 Internal 100 101 External 102103 PHM12755 17 Internal 104 105 External 106 107 PHM13879 18 Internal108 109 External 110 111 PHM4103 19 Internal 112 113 External 114 115PHM5363 20 Internal 116 117 External 118 119 PHM14751 21 Internal 120121 External 122 123 PHM16138 22 Internal 124 125 External 126 127PHM7877 23 Internal 128 129 External 130 131 PHM9518 24 Internal 132 133External 134 135 PHM7802 25 Internal 136 137 External 138 139 PHM2134 26Internal 140 141 External 142 143 PHM7808 27 Internal 144 145 External146 147 PHM9627 28 Internal 148 149 External 150 151 PHM13716 29Internal 152 153 External 154 155 PHM18731 30 Internal 156 157 External158 159 PHM2189 31 Internal 160 161 External 162 163 PHM7734 32 Internal164 165 External 166 167 PHM3323 33 Internal 168 169 External 170 171PHM4736 34 Internal 172 173 External 174 175 PHM6441 35 Internal 176 177External 178 179 PHM430 36 Internal 180 181 External 182 183 PHM12224 37Internal 184 185 External 186 187 PHM11904 38 Internal 188 189 External190 191 PHM6727 39 Internal 192 193 External 194 195 PHM4578 196Internal 215 216 External 214 217 PHM11186 197 Internal 219 220 External218 221 PHM14053 198 Internal 223 224 External 222 225 PHM16736 199Internal 227 228 External 226 229 PHM7844 200 Internal 231 232 External230 233 PHM8029 201 Internal 235 236 External 234 237 PHM2130 202Internal 239 240 External 238 241 PHM11754 203 Internal 243 244 External242 245 PHM1481 204 Internal 247 248 External 246 249 PHM15089 205Internal 251 252 External 250 253 PHM574 206 Internal 255 256 External254 257 PHM6427 207 Internal 259 260 External 258 261 PHM11125 208Internal 263 264 External 262 265 PHM13958 209 Internal 267 268 External266 269 PHM10468 210 Internal 271 272 External 270 273 PHM12706 211Internal 275 276 External 274 277 PHM405 212 Internal 279 280 External278 281 PHM12025 213 Internal 283 284 External 282 285

SEQ ID NOs:1-285 (See Table 1).

SEQ ID NOs:286-389 (See Table 2).

TABLE 2 Production Markers and Their Sequences Forward Reverse ProbeProbe Primer Primer Allele Allele Dye Dye 1 2 Marker Name SEQ ID NO:Sense 1 2 1 2 SEQ ID NO: PHM18693-9-U 286 287 Anti- T C Red Fam 288 289Sense PHM10786-11- 290 291 Sense T G Red Fam 292 293 U PHM10786-5-U 294295 Anti- C A Red Fam 296 297 Sense PHM10786-6-U 298 299 Anti- T C RedFam 300 301 Sense PHM8057-801- 302 303 Anti- T G Red Fam 304 305 U SensePHM4044-11-U 306 307 Sense T A Red Fam 308 309 PHM14080-16- 310 311Sense G A Red Fam 312 313 V PHM15089-10- 314 315 Sense G C Red Fam 316317 U PHM9364-6-U 318 319 Sense G A Red Fam 320 321 PHM201-16-U 322 323Anti- T C Red Fam 324 325 Sense PHM201-17-U 326 327 Sense C G Red Fam328 329 PHM4861-20-U 330 331 Sense T G Red Fam 332 333 PHM4861-21-U 334335 Anti- G A Red Fam 336 337 Sense PHM5421-5-V 338 339 Sense G T RedFam 340 341 PHM4115-35-U 342 343 Anti- G T Red Fam 344 345 SensePHM12521-18- 346 347 Sense T G Red Fam 348 349 U PHM12521-19- 350 351Sense G A Red Fam 352 353 U PHM12521-29- 354 355 Sense G A Red Fam 356357 U C00386-397-U 358 359 Sense T C Red Fam 360 361 PHM13418-18- 362363 Sense T C Fam Red 364 365 U PHM13418-10- 366 367 Sense C T Fam Red368 369 U PHM113-7-U 370 371 Anti- C T Fam Red 372 373 SensePHM10337-11- 374 375 Anti- T C Red Fam 376 377 U Sense PHM16736-8-V 378379 Sense T A Red Fam 380 381 PHM12025-48- 382 383 Anti- T C Red Fam 384385 U Sense PHM11186-16- 386 387 Sense T C Red Fam 388 389 V

DETAILED DESCRIPTION

The present invention provides allelic compositions in maize and methodsfor identifying and for selecting maize plants with favorable mechanicalstalk strength. Also within the scope of this invention are alleliccompositions and methods used to identify and to counter-select maizeplants that have decreased mechanical stalk strength. The followingdefinitions are provided as an aid to understand this invention.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus.

The term “altered mechanical stalk strength” refers to an increase or adecrease in the ability of maize plants to resist breakage as a resultof having a particular allele at a marker locus or a combination ofalleles at multiple marker loci.

An “amplicon” is a DNA fragment generated using the polymerase chainreaction.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR) and RNA polymerase based amplification(e.g., by transcription) methods.

The term “assemble” applies to BACs and their propensities for comingtogether to form contiguous stretches of DNA. A BAC “assembles” to acontig based on sequence alignment, if the BAC is sequenced, or via thealignment of its BAC fingerprint to the fingerprints of other BACs. Theassemblies can be found using the Maize Genome Browser, which ispublicly available on the internet.

An allele is “associated with” a trait when it is linked to it and whenthe presence of the allele is an indicator that the desired trait ortrait form will occur in a plant comprising the allele.

A “BAC”, or bacterial artificial chromosome, is a cloning vector derivedfrom the naturally occurring F factor of Escherichia coli. BACs canaccept large inserts of DNA sequence. In maize, a number of BACs, orbacterial artificial chromosomes, each containing a large insert ofmaize genomic DNA, have been assembled into contigs (overlappingcontiguous genetic fragments, or ‘contiguous DNA”).

“Backcrossing” refers to the process whereby hybrid progeny arerepeatedly crossed back to one of the parents. In a backcrossing scheme,the “donor” parent refers to the parental plant with the desired gene orlocus to be introgressed. The “recipient” parent (used one or moretimes) or “recurrent” parent (used two or more times) refers to theparental plant into which the gene or locus is being introgressed. Forexample, see Ragot, M. et al. (1995) Marker-assisted backcrossing: apractical example, in Techniques et Utilisations des MarqueursMoleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al.,(1994) Marker-assisted Selection in Backcross Breeding, Analysis ofMolecular Marker Data, pp. 41-43. The initial cross gives rise to the F1generation; the term “BC1” then refers to the second use of therecurrent parent, “BC2” refers to the third use of the recurrent parent,and so on.

A centimorgan (“cM”) is a unit of measure of recombination frequency.One cM is equal to a 1% chance that a marker at one genetic locus willbe separated from a marker at a second locus due to crossing over in asingle generation.

As used herein, the term “chromosomal interval” designates a contiguouslinear span of genomic DNA that resides in planta on a singlechromosome. The genetic elements or genes located on a singlechromosomal interval are physically linked. The size of a chromosomalinterval is not particularly limited. In some aspects, the geneticelements located within a single chromosomal interval are geneticallylinked, typically with a genetic recombination distance of, for example,less than or equal to 20 cM, or alternatively, less than or equal to 10cM. That is, two genetic elements within a single chromosomal intervalundergo recombination at a frequency of less than or equal to 20% or10%.

A “chromosome” can also be referred to as a “linkage group”.

The term “complement” refers to a nucleotide sequence that iscomplementary to a given nucleotide sequence, i.e. the sequences arerelated by the base-pairing rules.

The term “contiguous DNA” refers to overlapping contiguous geneticfragments.

The term “crossed” or “cross” means the fusion of gametes viapollination to produce progeny (e.g., cells, seeds or plants). The termencompasses both sexual crosses (the pollination of one plant byanother) and selfing (self-pollination, e.g., when the pollen and ovuleare from the same plant). The term “crossing” refers to the act offusing gametes via pollination to produce progeny.

Maize plants with “decreased mechanical stalk strength” are more proneto stalk lodging and have mechanically weaker stalks. The term“decreased” relates to the degree of physical strength and/or the degreeof resistance to breakage and is used to describe the effect onmechanical stalk strength when a particular allele is present or absent.

A plant referred to herein as “diploid” has two sets (genomes) ofchromosomes.

A plant referred to herein as a “doubled haploid” is developed bydoubling the haploid set of chromosomes. A doubled haploid plant isconsidered a homozygous plant.

An “elite line” is any line that has resulted from breeding andselection for superior agronomic performance.

A “favorable allele” is the allele at a particular locus that confers,or contributes to an agronomically desirable phenotype and that allowsthe identification of plants with that agronomically desirable phenotypee.g., increased mechanical stalk strength. A “favorable allele” of amarker is a marker allele that segregates with the favorable phenotype.

Maize plants with “favorable” mechanical stalk strength characteristicshave higher than average mechanical stalk strength and are less prone tostalk lodging. Maize plants with “unfavorable” mechanical stalk strengthcharacteristics have lower than average mechanical stalk strength andare more prone to stalk lodging.

An “unfavorable allele” of a marker is a marker allele that segregateswith the unfavorable plant phenotype, therefore providing the benefit ofidentifying plants that can be removed from a breeding program orplanting.

“Fragment” is intended to mean a portion of a nucleotide sequence.Fragments can be used as hybridization probes or PCR primers usingmethods disclosed herein.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form. For eachgenetic map, distances between loci are measured by the recombinationfrequencies between them, and recombinations between loci can bedetected using a variety of markers. A genetic map is a product of themapping population, types of markers used, and the polymorphic potentialof each marker between different populations. The order and geneticdistances between loci (e.g. markers) can differ from one genetic map toanother. For example, 10 cM on the internally derived genetic map (alsoreferred to herein as “PHB” for Pioneer Hi-Bred) is roughly equivalentto 25-30 cM on the IBM2 2005 neighbors frame map (a high resolution mapavailable on maizeGDB). However, information can be correlated from onemap to another using a general framework of common markers. One ofordinary skill in the art can use the framework of common markers toidentify the positions of markers and loci of interest on eachindividual genetic map. A comparison of marker positions between theinternally derived genetic map and the IBM2 neighbors genetic map forthe chromosome 5 QTL can be seen in Table 7.

“Genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis.

“Genome” refers to the total DNA, or the entire set of genes, carried bya chromosome or chromosome set.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci, as contrasted withthe observable trait (the phenotype). Genotype is defined by theallele(s) of one or more known loci that the individual has inheritedfrom its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells that can be cultured into a whole plant.

A plant referred to as “haploid” has a single set (genome) ofchromosomes.

A “haplotype” is the genotype of an individual at a plurality of geneticloci, i.e. a combination of alleles. Typically, the genetic locidescribed by a haplotype are physically and genetically linked, i.e., onthe same chromosome segment. The term “haplotype” can refer topolymorphisms at a particular locus, such as a single marker locus, orpolymorphisms at multiple loci along a chromosomal segment.

A “heterotic group” comprises a set of genotypes that perform well whencrossed with genotypes from a different heterotic group (Hallauer et al.(1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley(ed.) Corn and corn improvement). Inbred lines are classified intoheterotic groups, and are further subdivided into families within aheterotic group, based on several criteria such as pedigree, molecularmarker-based associations, and performance in hybrid combinations (Smithet al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely usedheterotic groups in the United States are referred to as “Iowa StiffStalk Synthetic” (BSSS) and “Lancaster” or “Lancaster Sure Crop”(sometimes referred to as NSS, or non-Stiff Stalk).

The term “heterozygous” means a genetic condition wherein differentalleles reside at corresponding loci on homologous chromosomes.

The term “homozygous” means a genetic condition wherein identicalalleles reside at corresponding loci on homologous chromosomes.

The term “hybrid” refers to the progeny obtained between the crossing ofat least two genetically dissimilar parents.

“Hybridization” or “nucleic acid hybridization” refers to the pairing ofcomplementary RNA and DNA strands as well as the pairing ofcomplementary DNA single strands.

The term “hybridize” means to form base pairs between complementaryregions of nucleic acid strands.

An “IBM genetic map” refers to any of following maps: IBM, IBM2, IBM2neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2 2005 neighbors, orIBM2 2005 neighbors frame. IBM genetic maps are based on a B73×Mo17population in which the progeny from the initial cross were random-matedfor multiple generations prior to constructing recombinant inbred linesfor mapping. Newer versions reflect the addition of genetic and BACmapped loci as well as enhanced map refinement due to the incorporationof information obtained from other genetic maps.

The term “inbred” refers to a line that has been bred for genetichomogeneity.

Maize plants with “increased mechanical stalk strength” are resistant tostalk lodging and have mechanically stronger stalks. The term“increased” relates to the degree of physical strength and/or the degreeof resistance to breakage and is used to describe the effect onmechanical stalk strength when a particular allele is present or absent.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an insertion relative to a second line, orthe second line may be referred to as having a deletion relative to thefirst line.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g., aselected allele of a marker, a QTL, a transgene, or the like. In anycase, offspring comprising the desired allele can be repeatedlybackcrossed to a line having a desired genetic background and selectedfor the desired allele, to result in the allele becoming fixed in aselected genetic background.

The process of “introgressing” is often referred to as “backcrossing”when the process is repeated two or more times.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is associated with another marker locus or someother locus (for example, a mechanical stalk strength locus). Thelinkage relationship between a molecular marker and a phenotype is givenas a “probability” or “adjusted probability”. Linkage can be expressedas a desired limit or range. For example, in some embodiments, anymarker is linked (genetically and physically) to any other marker whenthe markers are separated by less than 50, 40, 30, 25, 20, or 15 mapunits (or cM). In some aspects, it is advantageous to define a bracketedrange of linkage, for example, between 10 and 20 cM, between 10 and 30cM, or between 10 and 40 cM. The more closely a marker is linked to asecond locus, the better an indicator for the second locus that markerbecomes. Thus, “closely linked loci” such as a marker locus and a secondlocus display an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be“proximal to” each other. Since one cM is the distance between twomarkers that show a 1% recombination frequency, any marker is closelylinked (genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency (in the case ofco-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Markers that show linkagedisequilibrium are considered linked. Linked loci co-segregate more than50% of the time, e.g., from about 51% to about 100% of the time. Inother words, two markers that co-segregate have a recombinationfrequency of less than 50% (and by definition, are separated by lessthan 50 cM on the same linkage group.) As used herein, linkage can bebetween two markers, or alternatively between a marker and a phenotype.A marker locus can be “associated with” (linked to) a trait, e.g.,mechanical stalk strength. The degree of linkage of a molecular markerto a phenotypic trait is measured, e.g., as a statistical probability ofco-segregation of that molecular marker with the phenotype.

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor. Appl. Genet. 38:226-231 (1968). When r²=1, completeLD exists between the two marker loci, meaning that the markers have notbeen separated by recombination and have the same allele frequency.Values for r² above ⅓ indicate sufficiently strong LD to be useful formapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)).Hence, alleles are in linkage disequilibrium when r² values betweenpairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1.0.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

A “locus” is a position on a chromosome, e.g. where a gene or marker islocated.

The term “lodge” is synonymous with break. Hence, stalks that lodge arethose that break at a position along the stalk.

The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science255:803-804 (1992)) is used in interval mapping to describe the degreeof linkage between two marker loci. A LOD score of three between twomarkers indicates that linkage is 1000 times more likely than nolinkage, while a LOD score of two indicates that linkage is 100 timesmore likely than no linkage. LOD scores greater than or equal to two maybe used to detect linkage.

“Maize” refers to a plant of the Zea mays L. ssp. mays and is also knownas corn.

The term “maize plant” includes: whole maize plants, maize plant cells,maize plant protoplast, maize plant cell or maize tissue cultures fromwhich maize plants can be regenerated, maize plant calli, and maizeplant cells that are intact in maize plants or parts of maize plants,such as maize seeds, maize cobs, maize flowers, maize cotyledons, maizeleaves, maize stems, maize buds, maize roots, maize root tips, and thelike.

A “marker” is a nucleotide sequence or encoded product thereof (e.g., aprotein) used as a point of reference. A marker can be derived fromgenomic nucleotide sequence or from expressed nucleotide sequences(e.g., from a spliced RNA or a cDNA), or from an encoded polypeptide.The term also refers to nucleic acid sequences complementary to orflanking the marker sequences, such as nucleic acids used as probes orprimer pairs capable of amplifying the marker sequence.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, e.g., DNA sequencing, PCR-based sequence specific amplificationmethods, detection of restriction fragment length polymorphisms (RFLP),detection of isozyme markers, detection of polynucleotide polymorphismsby allele specific hybridization (ASH), detection of amplified variablesequences of the plant genome, detection of self-sustained sequencereplication, detection of simple sequence repeats (SSRs), detection ofsingle nucleotide polymorphisms (SNPs), or detection of amplifiedfragment length polymorphisms (AFLPs). Well established methods are alsoknown for the detection of expressed sequence tags (ESTs) and SSRmarkers derived from EST sequences and randomly amplified polymorphicDNA (RAPD).

A “marker allele”, alternatively an “allele of a marker locus”, canrefer to one of a plurality of polymorphic nucleotide sequences found ata marker locus in a population that is polymorphic for the marker locus.Marker alleles designated with a number, such as e.g. PHM3468 allele 2,represent the specific combination of alleles, also referred to as a“marker haplotype”, at that specific marker locus.

“Marker assisted selection” (of MAS) is a process by which individualplants are selected based on marker genotypes.

“Marker assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

A “marker locus” is a specific chromosome location in the genome of aspecies where a specific marker can be found. A marker locus can be usedto track the presence of a second linked locus, e.g., a linked locusthat encodes or contributes to expression of a phenotypic trait. Forexample, a marker locus can be used to monitor segregation of alleles ata locus, such as a QTL, that are genetically or physically linked to themarker locus.

A “marker probe” is a nucleic acid sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence, through nucleic acidhybridization. Marker probes comprising 30 or more contiguousnucleotides of the marker locus (“all or a portion” of the marker locussequence) may be used for nucleic acid hybridization. Alternatively, insome aspects, a marker probe refers to a probe of any type that is ableto distinguish (i.e., genotype) the particular allele that is present ata marker locus. Nucleic acids are “complementary” when they specifically“hybridize”, or pair, in solution, e.g., according to Watson-Crick basepairing rules.

“Mechanical stalk strength” refers to the physical strength of a maizestalk and its resistance to breakage (also known as “lodging”).

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably and refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A “nucleotide” is amonomeric unit from which DNA or RNA polymers are constructed, andconsists of a purine or pyrimidine base, a pentose, and a phosphoricacid group. Nucleotides (usually found in their 5′-monophosphate form)are referred to by their single letter designation as follows: “A” foradenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

The terms “phenotype”, or “phenotypic trait” or “trait” refers to one ormore trait of an organism. The phenotype can be observable to the nakedeye, or by any other means of evaluation known in the art, e.g.,microscopy, biochemical analysis, or an electromechanical assay. In somecases, a phenotype is directly controlled by a single gene or geneticlocus, i.e., a “single gene trait”. In other cases, a phenotype is theresult of several genes.

Each “PHM” marker represents two sets of primers that when used in anested PCR, amplify a specific piece of DNA. The external set is used inthe first round of PCR, after which the internal sequences are used fora second round of PCR on the products of the first round. This increasesthe specificity of the reaction. A “physical map” of the genome is a mapshowing the linear order of identifiable landmarks (including genes,markers, etc.) on chromosome DNA. However, in contrast to genetic maps,the distances between landmarks are absolute (for example, measured inbase pairs or isolated and overlapping contiguous genetic fragments) andnot based on genetic recombination.

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant.

A “polymorphism” is a variation in the DNA that is too common to be duemerely to new mutation. A polymorphism preferably has a frequency of atleast 1% in a population. A polymorphism can include a single nucleotidepolymorphism (SNP), a simple sequence repeat (SSR), or aninsertion/deletion polymorphism, also referred to herein as an “indel”.

The “probability value” or “p-value” is the statistical likelihood thatthe particular combination of a phenotype and the presence or absence ofa particular marker allele is random. Thus, the lower the probabilityscore, the greater the likelihood that a phenotype and a particularmarker will co-segregate. In some aspects, the probability score isconsidered “significant” or “nonsignificant”. In some embodiments, aprobability score of 0.05 (p=0.05, or a 5% probability) of randomassortment is considered a significant indication of co-segregation.However, an acceptable probability can be any probability of less than50% (p=0.5). For example, a significant probability can be less than0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05,less than 0.01, or less than 0.001.

A “production marker” or “production SNP marker” is a marker that hasbeen developed for high-throughput purposes. Production SNP markers weredeveloped for specific polymorphisms identified using PHM markers andthe nested PCR analysis. These production SNP markers were specificallydesigned for use with the Invader Plus® (Third Wave Technologies)platform.

The term “progeny” refers to the offspring generated from a cross.

A “progeny plant” is generated from a cross between two plants.

The term “quantitative trait locus” or “QTL” refers to a region of DNAthat is associated with the differential expression of a phenotypictrait in at least one genetic background, e.g., in at least one breedingpopulation. QTLs are closely linked to the gene or genes that underliethe trait in question.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. The reference sequence for a PHM marker is obtainedby genotyping a number of lines at the locus, aligning the nucleotidesequences in a sequence alignment program (e.g. Sequencher), and thenobtaining the consensus sequence of the alignment. Hence, a referencesequence identifies the polymorphisms in alleles at a locus. A referencesequence may not be a copy of an actual DNA sequence; however, it isuseful for designing primers and probes for actual polymorphisms in thelocus.

“Stalk lodging” refers to the breakage of the stalk. Stalk lodgingtypically occurs at or below the ear, but can occur at any positionalong the stalk.

A “three-point bend test” is an electromechanical system for evaluatingmechanical stalk strength. In this test, load can be applied tointernodes below the ear using an Instron™ machine, such as Model 4411(Instron Corporation, 100 Royall Street, Canton, Mass. 02021), or othercrushing device. The load needed to break the internode is used as ameasure of mechanical strength. The mechanical stalk strength valuesobtained from the three-point bend test have shown to be highlycorrelated to lodging scores that have been assigned based on fieldobservations.

A “topcross test” is a test performed on progeny derived by crossingeach parent with the same tester, usually a homozygous line. The parentbeing tested can be an open-pollinated variety, a cross, or an inbredline.

The phrase “under stringent conditions” refers to conditions under whicha probe or polynucleotide will hybridize to a specific nucleic acidsequence, typically in a complex mixture of nucleic acids, but toessentially no other sequences. Stringent conditions aresequence-dependent and will be different in different circumstances.

Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5-10° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength pH. The Tm is the temperature (under definedionic strength, pH, and nucleic acid concentration) at which 50% of theprobes complementary to the target hybridize to the target sequence atequilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium). Stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.01 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is at least about 30° C.for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C.for long probes (e.g., greater than 50 nucleotides). Stringentconditions may also be achieved with the addition of destabilizingagents such as formamide. For selective or specific hybridization, apositive signal is at least two times background, preferably 10 timesbackground hybridization. Exemplary stringent hybridization conditionsare often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or,5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDSat 65° C. For PCR, a temperature of about 36° C. is typical for lowstringency amplification, although annealing temperatures may varybetween about 32° C. and 48° C., depending on primer length. Additionalguidelines for determining hybridization parameters are provided innumerous references.

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Before describing the present invention in detail, it should beunderstood that this invention is not limited to particular embodiments.It also should be understood that the terminology used herein is for thepurpose of describing particular embodiments, and is not intended to belimiting. As used herein and in the appended claims, terms in thesingular and the singular forms “a”, “an” and “the”, for example,include plural referents unless the content clearly dictates otherwise.Thus, for example, reference to “plant”, “the plant” or “a plant” alsoincludes a plurality of plants. Depending on the context, use of theterm “plant” can also include genetically similar or identical progenyof that plant. The use of the term “a nucleic acid” optionally includesmany copies of that nucleic acid molecule.

Turning now to the embodiments:

Stalk Lodging and Mechanical Stalk Strength

Methods for identifying and for selecting maize plants with favorable orunfavorable mechanical stalk strength characteristics through thegenotyping of associated marker loci are provided. Mechanical stalkstrength in maize is an agronomically important trait, as increasedmechanical stalk strength enhances resistance to stalk lodging.

Stalk lodging, or stalk breakage, can occur at various developmentalstages. During vegetative growth, internodes are rapidly elongating,causing cell walls to thin, thereby making stalks more prone tobreakage. This type of breakage is called green snap, or brittle snap,and most often occurs between the vegetative stages of V5 and V8 andbetween the vegetative V12 stage and the reproductive R1 stage. Plantsthat lodge during the V5 to V8 stage usually do not recover, sincebreakage typically occurs below the growing point. Between thevegetative V12 stage and the reproductive R1 stage, stalks typicallybreak at nodes just below or above the ear. If the site of breakage isbelow the ear, ear development is severely impeded, resulting in nograin production. If the site of breakage is above the ear, limited orno grain production may still result, due to the lack of photosyntheticsurface area, which is required for supplying the developing ear(s) withnutrients.

Stalk lodging can also occur late season. As a maize plant matures, earweight increases, as does the load imposed on the stalk. The increasedload can cause the maize stalk to break, especially when additionalmechanical stresses, either biotic or abiotic, are imposed on the plant.If stalk breakage or lodging does occur, the ear may fall to the groundor to a height where harvest machinery cannot access the ear, therebyreducing yield. Alternatively, the proximity of the fallen ear to theground increases the probability of fungal spores being splashed on tothe ear, resulting in a loss of grain quality.

Maize plants with increased mechanical stalk strength, however, have agreater capacity to bear the weight of the ear and any appliedmechanical force. It is therefore desirable to identify and select maizeplants with increased mechanical stalk strength to prevent yield andgrain quality losses due to stalk lodging. It is also desirable toeliminate maize plants with decreased mechanical stalk strength frommaize breeding programs for the same purpose.

QTL Mapping

It has been recognized for quite some time that specific chromosomalloci (or intervals) in an organism's genome that correlate withparticular quantitative phenotypes, such as mechanical stalk strength,can be mapped genetically using markers. Such loci are termedquantitative trait loci, or QTL. The plant breeder can advantageouslyuse molecular markers to identify desired individuals by identifyingmarker alleles that show a statistically significant probability ofco-segregation with a desired phenotype, manifested as linkagedisequilibrium. By identifying a molecular marker or clusters ofmolecular markers that co-segregate with a quantitative trait, thebreeder is thus identifying a QTL. By identifying and selecting a markerallele (or desired alleles from multiple markers) that associates withthe desired phenotype, the plant breeder is able to rapidly select adesired phenotype by selecting for the proper molecular marker allele (aprocess called marker-assisted selection, or MAS). Such markers couldalso be used by breeders to design genotypes in silico and to practicewhole genome selection.

A variety of methods well known in the art are available for detectingmolecular markers or clusters of molecular markers that co-segregatewith a quantitative trait such as mechanical stalk strength. The basicidea underlying these methods is the detection of markers, for whichalternative genotypes (or alleles) have significantly different averagephenotypes. Thus, one makes a comparison among marker loci of themagnitude of difference among alternative genotypes (or alleles) or thelevel of significance of that difference. Trait genes are inferred to belocated nearest the marker(s) that have the greatest associatedgenotypic difference.

Two such methods used to detect QTLs are: 1) Population-based structuredassociation analysis and 2) Pedigree-based association analysis. In apopulation-based structured association analysis, lines are obtainedfrom pre-existing populations with multiple founders, e.g. elitebreeding lines. Population-based association analyses rely on the decayof linkage disequilibrium (LD) and the idea that in an unstructuredpopulation, only correlations between QTL and markers closely linked tothe QTL will remain after so many generations of random mating. Inreality, most pre-existing populations have population substructure.Thus, the use of a structured association approach helps to controlpopulation structure by allocating individuals to populations using dataobtained from markers randomly distributed across the genome, therebyminimizing disequilibrium due to population structure within theindividual populations (also called subpopulations). The phenotypicvalues are compared to the genotypes (alleles) at each marker locus foreach line in the subpopulation. A significant marker-trait associationindicates the close proximity between the marker locus and one or moregenetic loci that are involved in the expression of that trait.

The same principles underlie traditional linkage analysis; however, LDis generated by creating a population from a small number of founders.The founders are selected to maximize the level of polymorphism withinthe constructed population, and polymorphic sites are assessed for theirlevel of cosegregation with a given phenotype. A number of statisticalmethods have been used to identify significant marker-traitassociations. One such method is an interval mapping approach (Landerand Botstein, Genetics 121:185-199 (1989), in which each of manypositions along a genetic map (say at 1 cM intervals) is tested for thelikelihood that a gene controlling a trait of interest is located atthat position. The genotype/phenotype data are used to calculate foreach test position a LOD score (log of likelihood ratio). When the LODscore exceeds a threshold value, there is significant evidence for thelocation of a QTL at that position on the genetic map (which will fallbetween two particular marker loci).

The present invention provides QTLs that demonstrate statisticallysignificant co-segregation with mechanical stalk strength, as determinedby association analyses. Detection of these loci or additional linkedloci can be used in marker assisted maize breeding programs to produceplants with a favorable mechanical stalk strength phenotype or toeliminate plants with an unfavorable mechanical stalk strength phenotypefrom breeding programs or planting.

Markers Associated with Mechanical Stalk Strength

Markers associated with mechanical stalk strength are identified herein.

For the QTL identified on chromosome 5 (referred to herein as QTL5), themarker locus can be selected from any of the marker loci provided inTable 3 or Table 7, including the PHM markers, PHM201, PHM5421, PHM3468,PHM12521, and PHM10840, and the production SNP markers PHM201-16-U,PHM201-17-U, PHM4861-20-U, PHM4861-21-U, PHM5421-5-V, PHM4115-35-U,PHM12521-18-U, PHM12521-19-U, PHM12521-29-U, and C00386-397-U, as wellas any other marker linked to these QTL markers (linked markers can bedetermined from the MaizeGDB resource; see framework of markers in Table7).

For the QTL identified on chromosome 9 (referred to herein as QTL9), themarker locus can be selected from any of the marker loci provided inExample 1C, Table 5, or Table 9, including the PHM markers, PHM4578,PHM11186, PHM12025, PHM14053, PHM405, and PHM16736, and the productionSNP markers, PHM13418-18, PHM13418-10, PHM113-7, PHM10337-11-U,PHM16736-8-V, PHM12025-48, and PHM11186-16-V, as well as any othermarker linked to these QTL markers.

For one of the QTLs identified on chromosome 1 (referred to herein asQTL1A), the marker locus can be selected from any of the marker lociprovided in Example 1D, Table 5, or Table 9, including the PHM markers,PHM7844, PHM8029, PHM2130, and PHM574, and the production SNP markers,PHM18693-9-U, PHM10786-11-U, PHM10786-5-U, PHM10786-6-U, andPHM8057-801-U, as well as any other marker linked to these QTL markers.

For one of the QTLs identified on chromosome 1 (referred to herein asQTL1B), the marker locus can be selected from any of the QTL marker lociprovided in Example 1D, Table 5, or Table 9, including the PHM markers,PHM11754, PHM1481, PHM6427, and PHM15089, and the production SNPmarkers, PHM4044-11-U, PHM14080-16-V, PHM15089-10-U, and PHM9364-6-U, aswell as any other marker linked to these QTL markers.

For one of the QTLs identified on chromosome 1 (referred to herein asQTL1C), the marker locus can be selected from any of the QTL marker lociprovided in Example 1D, Table 5, or Table 9, including the PHM markers,PHM11125, PHM13958, PHM10468, and PHM12706, as well as any other markerlinked to these QTL markers.

Physical Map Locations of QTLs

The genetic elements or genes located on a contiguous linear span ofgenomic DNA on a single chromosome are physically linked.

For the QTL5 region, the two markers with the largest physical distancebetween them that still remain associated with the phenotype ofinterest, mechanical stalk strength, are PHM654 (reference sequence=SEQID NO:1) and PHM6727 (reference sequence=SEQ ID NO:39). PHM654 islocated on BAC c0216105, and PHM6727 is located on BAC c0117h02. Hence,these two BACs delineate the mechanical stalk strength QTL on the maizephysical map. Any BAC that assembles to the contiguous DNA between andincluding BAC c0216l05 and BAC c0117h02 can house marker loci that areassociated with the mechanical stalk strength trait. FIGS. 1A-I show thephysical map arrangement of the sequenced BACs that make up thecontiguous stretch of DNA between and including BAC c0216l05 and BACc0117h02. The gaps (represented by dotted lines) are not gaps in thecontiguous stretch of DNA per se but are areas where BACs that have notbeen sequenced assemble to the physical map.

An area on chromosome 9 defined by and including PHM4578 and PHM11186delineates the QTL9 region. Any polynucleotide that can hybridize to thecontiguous DNA between and including SEQ ID NO:196 (the referencesequence for PHM4578), or a nucleotide sequence that is 95% identical toSEQ ID NO:196 based on the Clustal V method of alignment, and SEQ IDNO:197 (the reference sequence for PHM11186), or a nucleotide sequencethat is 95% identical to SEQ ID NO:197 based on the Clustal V method ofalignment, and that is associated with mechanical stalk strength can beused as a marker for mechanical stalk strength. On the current B73physical map, PHM4578 is located on BACs c0478c20, c0414c21, andb0505j22, while PHM11186 is located on BACs c0475 m02 and b0197d12.

An area on chromosome 1 defined by and including PHM7844 and PHM8029delineates the QTL1A region. Any polynucleotide that can hybridize tothe contiguous DNA between and including SEQ ID NO:200 (the referencesequence for PHM7844), or a nucleotide sequence that is 95% identical toSEQ ID NO:200 based on the Clustal V method of alignment, and SEQ IDNO:201 (the reference sequence for PHM8029), or a nucleotide sequencethat is 95% identical to SEQ ID NO:201 based on the Clustal V method ofalignment, and that is associated with mechanical stalk strength can beused as a marker for mechanical stalk strength. On the current B73physical map, PHM7844 is located on BAC b0109 m14, while PHM8029 islocated on BAC c0230j20.

An area on chromosome 1 defined by and including PHM11754 and PHM1481delineates the QTL1B region. Any polynucleotide that can hybridize tothe contiguous DNA between and including SEQ ID NO:203 (the referencesequence for PHM11754), or a nucleotide sequence that is 95% identicalto SEQ ID NO:203 based on the Clustal V method of alignment, and SEQ IDNO:204 (the reference sequence for PHM1481), or a nucleotide sequencethat is 95% identical to SEQ ID NO:204 based on the Clustal V method ofalignment, and that is associated with mechanical stalk strength can beused as a marker for mechanical stalk strength. On the current B73physical map, PHM11754 is not located on a sequenced BAC, while PHM1481is located on BAC c0347b01.

An area on chromosome 1 defined by and including PHM11125 and PHM13958delineates the QTL1C region. Any polynucleotide that can hybridize tothe contiguous DNA between and including SEQ ID NO:208 (the referencesequence for PHM11125), or a nucleotide sequence that is 95% identicalto SEQ ID NO:208 based on the Clustal V method of alignment, and SEQ IDNO:209 (the reference sequence for PHM13958), or a nucleotide sequencethat is 95% identical to SEQ ID NO:209 based on the Clustal V method ofalignment, and that is associated with mechanical stalk strength can beused as a marker for mechanical stalk strength. On the current B73physical map, PHM11125 is located on BAC c0042p07, while PHM13958 islocated on BAC c0188c22.

Linkage Relationships

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or in centiMorgans (cM). The cM is a unit ofmeasure of genetic recombination frequency. One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a traitat another locus due to crossing over in a single generation (meaningthe traits segregate together 99% of the time). Because chromosomaldistance is approximately proportional to the frequency of crossing overevents between traits, there is an approximate physical distance thatcorrelates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, one cM is equal to a 1% chance that a marker locuswill be separated from another locus, due to crossing over in a singlegeneration.

The closer a marker is to a QTL marker, the more effective andadvantageous that marker is as an indicator for the desired trait.Closely linked loci display an inter-locus cross-over frequency of about10% or less, preferably about 9% or less, still more preferably about 8%or less, yet more preferably about 7% or less, still more preferablyabout 6% or less, yet more preferably about 5% or less, still morepreferably about 4% or less, yet more preferably about 3% or less, andstill more preferably about 2% or less. In highly preferred embodiments,the relevant loci (e.g., a marker locus and a target locus such as aQTL) display a recombination frequency of about 1% or less, e.g., about0.75% or less, more preferably about 0.5% or less, or yet morepreferably about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cMor less apart. Put another way, two loci that are localized to the samechromosome, and at such a distance that recombination between the twoloci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be“proximal to” each other.

Although particular marker alleles can show co-segregation with themechanical stalk strength phenotype, it is important to note that themarker locus is not necessarily part of the QTL locus responsible forthe expression of the mechanical stalk strength phenotype. For example,it is not a requirement that the marker polynucleotide sequence be partof a gene that imparts mechanical stalk strength (for example, be partof the gene open reading frame). The association between a specificmarker allele with either a favorable or unfavorable mechanical stalkstrength phenotype is due to the original “coupling” linkage phasebetween the marker allele and the QTL allele in the ancestral maize linefrom which the QTL allele originated. Eventually, with repeatedrecombination, crossing over events between the marker and QTL locus canchange this orientation. For this reason, the favorable marker allelemay change depending on the linkage phase that exists within theresistant parent used to create segregating populations. This does notchange the fact that the marker can be used to monitor segregation ofthe phenotype. It only changes which marker allele is consideredfavorable in a given segregating population.

For QTL5, markers identified in Table 3 or Table 7, as well as anymarker within 50 cM of the markers identified in Table 3 or Table 7, canbe used to predict mechanical stalk strength trait in a maize plant.This includes any marker within 50 cM of the PHM markers, PHM201,PHM5421, PHM3468, PHM12521, and PHM10840, and the production SNP markersPHM201-16-U, PHM201-17-U, PHM4861-20-U, PHM4861-21-U, PHM5421-5-V,PHM4115-35-U, PHM12521-18-U, PHM12521-19-U, PHM12521-29-U, andC00386-397-U. For QTL9, markers identified in Example 1C, Table 5, orTable 9, as well as any marker within 50 cM of the markers identified inExample 1C, Table 5, or Table 9, can be used to predict mechanical stalkstrength in a maize plant. This includes any marker within 50 cM of thePHM markers, PHM4578, PHM11186, PHM12025, PHM14053, PHM405, andPHM16736, and of the production SNP markers, PHM13418-18, PHM13418-10,PHM113-7, PHM10337-11-U, PHM16736-8-V, PHM12025-48, and PHM11186-16-V.

For QTL1A, markers identified in Example 1D, Table 5, or Table 9, aswell as any marker within 50 cM of the markers identified in Example 1D,Table 5, or Table 9, can be used to predict mechanical stalk strength ina maize plant. This includes any marker within 50 cM of PHM7844,PHM8029, PHM2130, or PHM574, and of the production SNP markers,PHM18693-9-U, PHM10786-11-U, PHM10786-5-U, PHM10786-6-U, andPHM8057-801-U.

For QTL1B, markers identified in Example 1D, Table 5, or Table 9, aswell as any marker within 50 cM of the markers identified in Example 1D,Table 5, or Table 9, can be used to predict mechanical stalk strength ina maize plant. This includes any marker within 50 cM of the PHM markers,PHM11754, PHM1481, PHM6427, and PHM15089, and of the production SNPmarkers, PHM4044-11-U, PHM14080-16-V, PHM15089-10-U, and PHM9364-6-U.

For QTL1C, markers identified in Example 1D, Table 5, or Table 9, aswell as any marker within 50 cM of the markers identified in Example 1D,Table 5, or Table 9, can be used to predict mechanical stalk strength ina maize plant. This includes any marker within 50 cM of the PHM markers,PHM11125, PHM13958, PHM10468, and PHM12706.

Chromosomal Intervals

Chromosomal intervals that correlate with mechanical stalk strength areprovided. A variety of methods well known in the art are available foridentifying chromosomal intervals. The boundaries of such chromosomalintervals are drawn to encompass markers that will be linked to one ormore QTL. In other words, the chromosomal interval is drawn such thatany marker that lies within that interval (including the terminalmarkers that define the boundaries of the interval) can be used as amarker for mechanical stalk strength. Each interval comprises at leastone QTL, and furthermore, may indeed comprise more than one QTL. Closeproximity of multiple QTL in the same interval may obfuscate thecorrelation of a particular marker with a particular QTL, as one markermay demonstrate linkage to more than one QTL. Conversely, e.g., if twomarkers in close proximity show co-segregation with the desiredphenotypic trait, it is sometimes unclear if each of those markersidentify the same QTL or two different QTL. Regardless, knowledge of howmany QTL are in a particular interval is not necessary to make orpractice the invention.

Each of the intervals described shows a clustering of markers thatco-segregate with mechanical stalk strength. This clustering of markersoccurs in relatively small domains on the linkage groups, indicating thepresence of one or more QTL in those chromosome regions. QTL intervalswere drawn to encompass the markers that co-segregate with mechanicalstalk strength. The intervals are defined by the markers on theirtermini, where the interval encompasses markers that map within theinterval, whether known or unknown, as well as the markers that definethe termini.

For QTL5, any marker located within any of the following intervals findsuse as a marker for mechanical stalk strength. These intervals include:

-   -   (i) PHM654 and PHM6727;    -   (ii) PHM12632 and PHM3323;    -   (iii) PHM201 and PHM3323; and    -   (iv) PHM201 and PHM3468.

PHM654 and PHM6727 are separated by ˜25 cM on the internally-derivedgenetic map and define a chromosomal interval encompassing a cluster ofmarkers that co-segregate with mechanical stalk strength in the NSSsubpopulation at a p-level of ≦0.01 (FIG. 2C). PHM12632 and PHM3323,separated by ˜9 cM on the internally-derived genetic map, define achromosomal interval encompassing a cluster of markers that co-segregatewith mechanical stalk strength in the NSS subpopulation at a p-level of≦0.001 (FIG. 2D). PHM201 and PHM3323, separated by ˜9 cM on theinternally-derived genetic map, define a chromosomal intervalencompassing a cluster of markers that co-segregate with mechanicalstalk strength in the NSS subpopulation at a p-level of ≦0.0001 (FIG.2E).

For QTL9, any marker located within any of the following intervals findsuse as a marker for mechanical stalk strength:

-   -   (i) PHM4578 and PHM11186 and    -   (ii) PHM14053 and PHM16736.

PHM4578 and PHM11186, separated by ˜3.5 cM on the internally-derivedgenetic map, define a chromosomal interval encompassing a cluster ofmarkers that co-segregate with mechanical stalk strength in the NSSsubpopulation at a p-level of ≦0.01. PHM14053 and PHM16736, separated by˜1.5 cM on the internally-derived genetic map, define a chromosomalinterval encompassing a cluster of markers that co-segregate withmechanical stalk strength in the NSS subpopulation at a p-level of≦0.001.

For QTL1A, any marker located within any of the following intervalsfinds use as a marker for mechanical stalk strength:

-   -   (i) PHM7844 and PHM8029 and    -   (ii) PHM7844 and PHM574.

PHM7844 and PHM8029, separated by ˜16 cM on the internally-derivedgenetic map, define a chromosomal interval encompassing a cluster ofmarkers that co-segregate with mechanical stalk strength in the NSSsubpopulation at a p-level of ≦0.01. PHM7844 and PHM574, separated by˜6.5 cM on the internally-derived genetic map, define a chromosomalinterval encompassing a cluster of markers that co-segregate withmechanical stalk strength in the NSS subpopulation at a p-level of≦0.001.

For QTL1B, any marker located within any of the following intervalsfinds use as a marker for mechanical stalk strength:

-   -   (i) PHM11754 and PHM1481 and    -   (ii) PHM6427 and PHM1481.

PHM11754 and PHM1481, separated by ˜21 cM on the internally-derivedgenetic map, define a chromosomal interval encompassing a cluster ofmarkers that co-segregate with mechanical stalk strength in the NSSsubpopulation at a p-level of ≦0.01. PHM6427 and PHM1481, separated by˜21 cM on the internally-derived genetic map, define a chromosomalinterval encompassing a cluster of markers that co-segregate withmechanical stalk strength in the NSS subpopulation at a p-level of≦0.001.

For QTL1C, any marker located within any of the following intervalsfinds use as a marker for mechanical stalk strength:

-   -   (i) PHM11125 and PHM13958 and    -   (ii) PHM10468 and PHM13958.

PHM11125 and PHM13958, separated by ˜15 cM on the internally-derivedgenetic map, define a chromosomal interval encompassing a cluster ofmarkers that co-segregate with mechanical stalk strength in the NSSsubpopulation at a p-level of ≦0.01. PHM10468 and PHM13958, separated by˜12 cM on the internally-derived genetic map, define a chromosomalinterval encompassing a cluster of markers that co-segregate withmechanical stalk strength in the NSS subpopulation at a p-level of≦0.001.

Chromosomal intervals can also be defined by markers that are in linkagedisequilibrium with a known QTL marker, and r² is a common measure oflinkage disequilibrium (LD) in the context of association studies. Forexample, if the r² value of LD between a chromosome 5 marker locus lyingwithin the interval of PHM654 and PHM6727 and any of the chromosome 5QTL markers identified in Table 3 or Table 7 is greater than ⅓ (Ardlieet al., Nature Reviews Genetics 3:299-309 (2002)), the loci are inlinkage disequilibrium.

Marker Alleles and Haplotypic Combinations

A marker of the invention can also be a combination of particularalleles at one or more marker loci, otherwise known as a haplotype. Thealleles described below could be used in combination to identify andselect for maize plants with mechanical stalk characteristics.

Favorable SNP alleles at QTL5 marker loci have been identified hereinand include: a “G” at position 314 in SEQ ID NO:12 (PHM5421), a “G” atposition 93 in SEQ ID NO:15 (PHM3468), a “T” at position 101 in SEQ IDNO:15 (PHM3468), a “G” at position 245 in SEQ ID NO:15 (PHM3468), a “T”at position 101 in SEQ ID NO:14 (PHM12521), an “A” at position 186 inSEQ ID NO:14 (PHM12521), a “C” at position 37 in SEQ ID NO:16(PHM10840), an “A” at position 240 in SEQ ID NO:16 (PHM10840, and a “C”at position 315 in SEQ ID NO:16 (PHM10840), a “C” at PHM201-16-U, a “C”or “G” at PHM201-17-U, a “T” or “G” at PHM4861-20-U, an “A” atPHM4861-21-U, a ‘G” at PHM5421-5-V, a “G” or “T” at PHM4115-35-U, a “T”at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, and a“C” at C00386-397-U.

Favorable SNP alleles at QTL9 marker loci have been identified hereinand include: a “C” at position 225 in SEQ ID NO:199 (PHM16736), a “T” atposition 326 in SEQ ID NO:199 (PHM16736), an “A” at position 422 in SEQID NO:199 (PHM16736), a “C” at position 193 in SEQ ID NO:198 (PHM14053),a “C” at position 341 in SEQ ID NO:198 (PHM14053), a “C” at position 386in SEQ ID NO:198 (PHM14053), a “T” at position 374 in SEQ ID NO:212(PHM405), a “C” at position 216 in SEQ ID NO:213 (PHM12025), a “C” atPHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” atPHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T”at PHM11186-16-V.

Favorable SNP alleles at QTL1A marker loci have been identified hereinand include: a “T” at position 75 of SEQ ID NO:202 (PHM2130), an “A” atposition 170 of SEQ ID NO:202 (PHM2130), a “C” at position 179 of SEQ IDNO:202 (PHM2130), a “G” at position 358 of SEQ ID NO:202 (PHM2130), a“T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C’ at PHM10786-5-U, a“T” at PHM10786-6-U, and a “G” at PHM8057-801-U.

Unfavorable SNP alleles at QTL5 marker loci have been identified hereinand include: a “C” at position 132 in SEQ ID NO:10 (PHM201) and an “A”at position 230 in SEQ ID NO:10 (PHM201).

Unfavorable SNP alleles at QTL1B marker loci have been identified hereinand include: a “G” at position 284 in SEQ ID NO:205 (PHM15089), a “T” atPHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G”at PHM9364-6-U.

An unfavorable SNP allele at a QTL1C marker locus has been identifiedherein, a “C” at position 322 of SEQ ID NO:210.

The skilled artisan would expect that there might be additionalpolymorphic sites at marker loci in and around the QTL markersidentified herein, wherein one or more polymorphic sites is in highlinkage disequilibrium (LD) with an allele at one or more of thepolymorphic sites in the haplotype. Two particular alleles at differentpolymorphic sites are said to be in LD if the presence of the allele atone of the sites tends to predict the presence of the allele at theother site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).

Marker Assisted Selection

Methods for marker assisted selection (MAS), in which phenotypes areselected based on marker genotypes, are also provided. To perform MAS, anucleic acid corresponding to the marker nucleic acid allele is detectedin a biological sample from a plant to be selected. This detection cantake the form of hybridization of a probe nucleic acid to a markerallele or amplicon thereof, e.g., using allele-specific hybridization,Southern analysis, northern analysis, in situ hybridization,hybridization of primers followed by PCR amplification of a region ofthe marker, DNA sequencing of a PCR amplification product, or the like.The procedures used to detect marker alleles are known to one ofordinary skill in the art. After the presence (or absence) of aparticular marker allele in the biological sample is verified, the plantis selected and is crossed to a second plant, e.g. a maize plant from anelite line. The progeny plants produced by the cross can be evaluatedfor that specific marker allele, and only those progeny plants that havethe desired marker allele will be chosen.

Maize plant breeders desire combinations of desired genetic loci, suchas those marker alleles associated with increased mechanical stalkstrength, with genes for high yield and other desirable traits todevelop improved maize varieties. Screening large numbers of samples bynon-molecular methods (e.g., trait evaluation in maize plants) can beexpensive, time consuming, and unreliable. Use of the polymorphicmarkers described herein, when genetically-linked to mechanical stalkstrength loci, provide an effective method for selecting varieties withincreased mechanical stalk strength in breeding programs. For example,one advantage of marker-assisted selection over field evaluations formechanical stalk strength is that MAS can be done at any time of year,regardless of the growing season. Moreover, environmental effects arelargely irrelevant to marker-assisted selection.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents or parentlines. Backcrossing is usually done for the purpose of introgressing oneor a few loci from a donor parent (e.g., a parent comprising desirablemechanical stalk strength marker loci) into an otherwise desirablegenetic background from the recurrent parent (e.g., an otherwise highyielding maize line). The more cycles of backcrossing that are done, thegreater the genetic contribution of the recurrent parent to theresulting introgressed variety. This is often necessary, because plantsmay be otherwise undesirable, e.g., due to low yield, low fecundity, orthe like. In contrast, strains which are the result of intensivebreeding programs may have excellent yield, fecundity or the like,merely being deficient in one desired trait such as mechanical stalkstrength.

One application of MAS is to use the markers to increase the efficiencyof an introgression or backcrossing effort aimed at introducing anincreased mechanical stalk strength QTL into a desired (typically highyielding) background. In marker assisted backcrossing of specificmarkers (and associated QTL) from a donor source, e.g., to an elite orexotic genetic background, one selects among backcross progeny for thedonor trait and then uses repeated backcrossing to the elite or exoticline to reconstitute as much of the elite/exotic background's genome aspossible.

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with the mechanicalstalk strength trait. Such markers are presumed to map near a gene orgenes that give the plant its mechanical stalk strength phenotype, andare considered indicators for the desired trait, and hence, are termedQTL markers. Plants are tested for the presence of a desired allele inthe QTL marker, and plants containing a desired genotype at one or moreloci are expected to transfer the desired genotype, along with a desiredphenotype, to their progeny.

The markers and QTL intervals presented herein find use in MAS to selectmaize plants or germplasm for mechanical stalk strength characteristics.Methods for selection involve obtaining DNA accessible for analysis,detecting the presence or absence of either an identified marker alleleor an unknown marker allele that is linked to and associated with anidentified marker allele, and then selecting the maize plant orgermplasm based on the allele detected.

Marker alleles that can be detected include: a “G” at PHM3468.1, a “T”at PHM3468.4, a “G” at PHM3468.18, a “T” at PHM12521.12, a “C” atPHM10840.105, an “A” at PHM10840.118, a “C” at PHM10840.130, a “C” atPHM16736.6, an “A” at PHM16736.14, a “C” at PHM14053.7, a “C” atPHM14053.8, a “C” at PHM14053.14, a “T” at PHM405.35, a “C” atPHM12025.26, a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C” atPHM10786-5-U, a “T” at PHM10786-6-U, a “G” at PHM8057-801-U, a “C” atPHM201-16-U, a “C” at PHM201-17-U, a “G” at PHM201-17-U, a “T” atPHM4861-20-U, a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” atPHM5421-5-V, a “G” at PHM4115-35-U, a “T” at PHM4115-35-U, a “T” atPHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” atC00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” atPHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” atPHM12025-48, a “T” at PHM11186-16-V, and any marker allele associatedwith and linked to any of the marker alleles listed above by 30, 25, 20,15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, or 0.1 cM. The maize plant or germplasm that has any of thesemarker alleles can be selected.

Marker alleles that can be detected can also include: a “T” atPHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” atPHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” atPHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” atPHM14080-16-V, a “C” at PHM15089-10-U, a “G” at PHM9364-6-U, and anymarker allele associated with and linked to any of the marker alleleslisted above by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. The maize plant or germplasm thatdoes not have any of these marker alleles can be selected.

Methods for selecting maize plants with mechanical stalk strengthcharacteristics can also involve detecting alleles at one or more markerloci lying within specific chromosomal intervals or assaying at leastone marker locus within a specific interval in which the marker locus isassociated with mechanical stalk strength.

For instance, a maize plant having at least one allele of a marker locuslying within any of the following QTL5 intervals:

(i) PHM654 and PHM6727;

(ii) PHM12632 and PHM3323;

(iii) PHM201 and PHM3323; and

(iv) PHM201 and PHM3468, wherein the allele is associated with increasedmechanical stalk strength, can be crossed to another maize plant, and aprogeny plant arising from the cross can be evaluated for the markerallele that is associated with increased mechanical stalk strength andthen selected if it possesses the marker allele.

Methods can also include assaying at least one marker locus in the maizeplant. The marker can lie within any of the following chromosomalintervals comprising and flanked by:

(1) PHM7844 and PHM8029;

(2) PHM7844 and PHM574;

(3) PHM11754 and PHM1481;

(4) PHM6427 and PHM1481;

(5) PHM11125 and PHM13958;

(6) PHM10468 and PHM13958;

(7) PHM4578 and PHM11186; or

(8) PHM14053 and PHM16736; and the marker locus is associated withmechanical stalk strength. Either a maize plant that possesses afavorable allele or a maize plant that does not possess an unfavorableallele could then be selected.

Phenotypic Assessment of Mechanical Stalk Strength

Any method known in the art can be used to evaluate mechanical stalkstrength. Some methods involve the measurement of stalk diameter or dryweight per plant, while others can utilize an Instron™ machine or othersimilar crushing device to assess the load needed to break a stalk. Thethree point bend test is often used in conjunction with an Instron™machine or other similar crushing device, and mechanical stalk strengthvalues obtained from the three-point bend test have shown to be highlycorrelated to lodging scores assigned based on field observations. Stillanother method can involve the use of a stalk-penetrating device.

In addition, any method that uses a device to accurately reproduce windforces, in order to select plants with increased mechanical stalkstrength in the field, can be utilized for the characterization ofmechanical stalk strength in maize plants and for the identification offavorable and/or undesirable quantitative trait loci (QTLs) associatedwith mechanical stalk strength. A device and method used to screen forselected wind-resistance traits in maize, including stalk strength, aredescribed in patent application US2007/0125155 (published Jun. 6, 2007).When this device and method are used, the unit of measure is the numberor percentage of plants that have lodged, or broken, stalks (or,alternatively, the number or percentage of plants that do not lodge).

EXAMPLES

The following examples are offered to illustrate, but not to limit, theappended claims. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that personsskilled in the art will recognize various reagents or parameters thatcan be altered without departing from the spirit of the invention or thescope of the appended claims.

Example 1A QTL Detection: Association Mapping Analysis

An association mapping strategy was undertaken to identify markersassociated with mechanical stalk strength in maize. In this associationanalysis, a collection of 599 maize lines was analyzed by DNA sequencingat 4000-10000 genes (genetic loci). The lines encompassed elitegermplasm, commercially released cultivars, and other public varieties.

Phenotypic scores were obtained from 189 individuals in the collection.Mechanical stalk strength was measured using an Instron™ machine, Model4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021), anda three point bend test, with the force applied between nodes 3 and 4below the ear. The score was the load applied to break the internode, orthe weight in kg applied to the internode at the yielding, or breaking,point. Data collection was typically done in one scoring after floweringtime (at end of season), and an average score for each line was assignedbased on data accumulated over multiple locations. Mechanical stalkstrength values for the 189 lines varied from 4.208 to 19.569 kg atyield point. Plants with high scores have greater relative mechanicalstalk strength.

The phenotypic scores and marker information for each of the 189 lineswas input into the association analysis. A structure-based associationanalysis was conducted using standard association mapping methods, wherethe population structure is controlled using marker data. Themodel-based cluster analysis software, Structure, developed by Pritchardet al., (Genetics 155:945-959 (2000)) was used with haplotype data for880 elite maize inbreds at two hundred markers to estimate admixturecoefficients and assign the inbreds to seven subpopulations. Thisreduces the occurrence of false positives that can arise due to theeffect of population structure on association mapping statistics.Kuiper's statistic for testing whether two distributions are the samewas used to test a given marker for association between haplotype andphenotype in a given subpopulation (Press et al., Numerical Recipes inC, second edition, Cambridge University Press, NY (2002)).

QTLs of interest were identified based on the results of the associationanalyses. These QTLs are described in examples 1B-D and are shown inFIGS. 2-4. In FIGS. 2-4, positions are given in cM, with position zerobeing the first (most distal from the centromere) marker known at thebeginning of the chromosome. These map positions are not absolute andrepresent an estimate of map position based on the internally derivedgenetic map (PHB).

Example 1B Identification of QTL5

A peak of significant marker-trait associations was identified onchromosome 5 (FIG. 2A). Further analysis at this peak revealed itsoccurrence in one of the seven subpopulations (FIG. 2B), a non-stiffstalk (NSS) group. Table 3 provides a listing of the chromosome 5markers significantly associated with the mechanical stalk strengthphenotype at the p≦0.01 level in this NSS subpopulation, representing aninterval of ˜25 cM on the internally derived genetic map. Thischromosomal interval is delineated by and includes markers PHM654 atposition 64.21 (p=0.007) and PHM6727 at position 89.62 (p=0.0031) (FIG.2C). Multiple markers on chromosome 5 are significantly associated withmechanical stalk strength at the p≦0.001 level, identifying an intervaldelineated by and including markers PHM12632 at position 71.5 (p=0.0003)and PHM3323 at position 80.89 (p=0.001) (FIG. 2D); these markers arenoted with an asterisk in Table 3. In addition, multiple markers onchromosome 5 are significantly associated with mechanical stalk strengthat the p≦0.0001 level, identifying an interval delineated by andincluding markers PHM201 at position 71.5 (p=0.0003) and PHM3323 atposition 80.89 (p=0.001) (FIG. 2E). The most associated markers arePHM201 at position 71.57 (p=0.00001), PHM5421 at position 72.15(p=0.0000052), and PHM3468 at position 76.53 (p=0.000064).

TABLE 3 Chromosome 5 markers significantly associated with mechanicalstalk strength at p ≦ 0.01 in the NSS subpopulation Marker Relative PHBmap Locus position (cM) P-Value  PHM654 64.21 0.007  PHM111 65.11 0.003 PHM10100 66.69 0.0023  PHM7357 67.42 0.0056  PHM5349 67.59 0.0018 PHM4167 69.19 0.0023  PHM14947 70.29 0.0018 *PHM12632 71.5 3.00E−04*PHM201 71.57 1.00E−05 *PHM4861 71.57 1.00E−04 *PHM5421 72.15 5.20E−06*PHM4115 73.37 5.00E−04 *PHM12521 74.86 1.00E−04 *PHM3468 76.53 6.40E−05*PHM10840 76.53 1.00E−04 *PHM12755 76.82 2.00E−04  PHM13879 77.01 0.0016 PHM4103 77.29 0.0079 *PHM5363 77.66 9.00E−04  PHM14751 77.84 0.0039 PHM16138 78.02 0.0012  PHM7877 78.86 0.0029  PHM9518 78.89 0.005 PHM7802 79.48 0.0047 *PHM2134 79.53 3.00E−04 *PHM7808 79.6 4.00E−04*PHM9627 79.6 0.001  PHM13716 79.6 0.0073  PHM18731 79.72 0.0019 PHM2189 80.36 0.0018 *PHM7734 80.78 1.20E−04 *PHM3323 80.89 1.00E−04 PHM12224 89.17 0.0026  PHM11904 89.45 0.0096  PHM6727 89.62 0.0031*Markers associated with mechanical stalk strength at p ≦ 0.001

There were 60 lines assigned by the model-based cluster analysissoftware, Structure, to the NSS subpopulation in which the QTL formechanical stalk strength were detected. The lines can be sorted byphenotype and can be assessed at the following marker loci: PHM201,PHM5421, and PHM3468. The phenotype and marker allele data for all 60lines at the following marker loci: PHM201, PHM5421, and PHM3468, areshown in Table 4. (For the PHM marker alleles, see FIG. 5.)

TABLE 4 Phenotype and marker allele data (PHM201, PHM5421, and PHM3468)for lines in NSS subpopulation Line Phenotype PHM201 PHM5421 PHM3468PH07H 19.569 2 2 8 PH14E 15.29 2 2 8 PH891 14.863 2 2 8 PHRF5 14.854 1 32 PH1AA 13.738 1 3 2 PH2V7 13.667 2 2 8 PHAP9 13.536 2 2 8 PH2FT 13.3552 2 8 PH1GC 12.48 4 2 8 PH589 12.432 1 3 2 PHDG1 12.421 4 2 8 PHG4412.335 2 2 NA PH2T6 11.993 2 2 8 PH1TB 11.983 2 2 8 PH806 11.783 2 2 8PHW89 11.683 2 2 8 PHM10 11.629 1 3 2 PHK42 11.492 1 3 2 PH7JD 11.311 22 8 PH8CW 11.22 1 3 2 PH81B 11.164 1 NA 2 PH8KF 10.871 2 2 NA PHJ9010.869 5 2 8 PH1CP 10.799 2 NA 8 PH8KG 10.531 2 2 8 PHR31 10.447 1 3 2PH3KP 10.206 5 2 8 PH1B5 10.148 2 2 8 PH0HR 10.142 2 NA 8 PHNG2 10.069 22 8 PHRF1 9.956 2 2 6 PHH93 9.919 1 3 2 PH7C8 9.88 1 3 2 PH24E 9.675 1 32 PH7DD 9.632 1 3 2 PH5HP 9.474 1 3 2 PH7CP 9.345 1 3 2 PHTE7 9.297 1 32 PH1N8 9.286 1 3 8 PHDP0 9.204 1 3 2 PH1W0 8.985 1 3 2 PHPP8 8.895 1 32 PH16M 8.855 1 3 2 PHK74 8.597 1 3 2 PH1G5S 8.468 1 3 2 PHG29 8.432 1 32 PHP55 8.317 1 3 2 PHN82 8.224 1 NA 2 PH0N7 8.182 1 3 2 PH3MW 8.167 1 36 PH05N 8.156 1 3 2 PH1G5R 8.014 2 2 8 PH23D 7.913 1 3 2 PH51K 7.629 1 32 PH7CM 7.596 1 3 6 PH1B8 7.503 1 3 6 PHG50 7.435 4 NA 8 PHKW3 7.143 1 36 PHACJ 6.764 1 3 6 PH3N0 6.629 1 3 6

Further analyses can be performed using marker data from PHM201 andPHM5421. Five lines have missing marker data, and four lines had ahaplotype that did not occur with a frequency of at least 10%. Thus,thirty three lines had the haplotype consisting of allele 1 at PHM201and allele 3 at PHM5421 (designated as “1-3”). The average phenotypicscore for the thirty three lines with haplotype 1-3 was 9.326 kg atyield point with a standard error of 0.107. The remaining eighteen lineshad the haplotype consisting of allele 2 at PHM201 and allele 2 atPHM5421 (designated as “2-2”). The average phenotypic score for theeighteen lines with haplotype 2-2 was 12.275 kg at yield point with astandard error of 0.370. A depiction of these results can be seen inFIG. 6A.

The allelic variation at PHM3468 can also be an indicator of phenotype.Of the 60 lines, two had missing marker data. Seven lines had markerallele 6 at PHM3468 and an average phenotypic score of 7.680 kg at yieldpoint with a standard error of 0.183. Twenty eight lines had markerallele 2 at PHM3468 and an average phenotypic score of 9.788 kg at yieldpoint with a standard error of 0.113. The remaining twenty three lineshad marker allele 8 at PHM3468 and an average phenotypic score of 11.801kg at yield point with a standard error of 0.289. A depiction of theseresults can be seen in FIG. 6B.

Thus, allele 1 at PHM201, allele 3 at PHM5421, allele 6 at PHM3468, andthe haplotype consisting of allele 1 at PHM201, allele 3 at PHM5421, andallele 6 at PHM3468 are associated with decreased mechanical stalkstrength, while allele 2 at PHM201, allele 2 at PHM5421, allele 8 atPHM3468, and the haplotype consisting of allele 2 at PHM201, allele 2 atPHM5421, and allele 8 at PHM3468 are associated with increasedmechanical stalk strength.

Example 1C Identification of QTL9

A peak of significant marker-trait associations was identified onchromosome 9 (FIG. 3) in the same non-stiff stalk (NSS) group in whichthe chromosome 5 QTL was identified. Chromosome 9 markers associatedwith mechanical stalk strength at p≦0.01 lie within a ˜3.5 cMchromosomal interval delineated by and including markers PHM4578 atposition 178.04 (p=0.0034) and PHM11186 at position 181.5 (p=0.0074),while markers associated with mechanical stalk strength at p≦0.001 liewithin a ˜1.5 cM chromosomal interval delineated by and includingmarkers PHM14053 at position 179.85 (p=0.001) and PHM16736 at position181.19 (p=4.00E-04). The top associated markers also include: PHM12025at position 179.78 (p=0.0015) and PHM405 at position 180.29 (p=0.0024).

Example 1D Identification of QTL1A, QTL1B, and QTL1C

Three peaks of significant marker-trait associations were identified onchromosome 1 (FIG. 4) in the same non-stiff stalk (NSS) group in whichthe chromosome 5 and 9 QTLs were identified.

In the first chromosome 1 interval, denoted as QTL1A, markers associatedwith mechanical stalk strength at p≦0.01 lie within a ˜16 cM chromosomalinterval delineated by and including markers PHM7844 at position 104.55(p=0.001) and PHM8029 at position 120.44 (p=0.0087), while markersassociated with mechanical stalk strength at p≦0.001 lie within a ˜6.5cM chromosomal interval delineated by and including markers PHM7844 atposition 104.55 (p=0.001) and PHM574 at position 111.02 (p=6.00E-04).The marker most significantly associated with mechanical stalk strengthis PHM2130 at position 107.69 (p=1.00E-04).

In the second chromosome 1 interval, denoted as QTL1B, markersassociated with mechanical stalk strength at p≦0.01 lie within a ˜21 cMchromosomal interval delineated by and including markers PHM11754 atposition 125.26 (p=0.0089) and PHM1481 at position 146.41 (p=2.00E-04),while markers associated with mechanical stalk strength at p≦0.001 liewithin a ˜21 cM chromosomal interval delineated by and including markersPHM6427 at position 125.63 (p=5.00E-04) and PHM1481 at position 146.41(p=2.00E-04). The marker most significantly associated with mechanicalstalk strength is PHM15089 at position 133.73 (p=4.00E-05).

In the third chromosome 1 interval, denoted as QTL1C, markers associatedwith mechanical stalk strength at p≦0.01 lie within a ˜15 cM chromosomalinterval delineated by and including markers PHM11125 at position 184.03(p=0.0019) and PHM13958 at position 198.8 (p=6.00E-04), while markersassociated with mechanical stalk strength at p≦0.001 lie within a ˜12 cMchromosomal interval delineated by and including markers PHM10468 atposition 187.27 (p=4.00E-04) and PHM13958 at position 198.8(p=6.00E-04). One marker significantly associated with mechanical stalkstrength is PHM12706 at position 191.11 (p=1.40E-04).

Example 1E Identification of Favorable and Unfavorable Marker Alleles

There were 60 lines assigned by the model-based cluster analysissoftware, Structure, to the NSS subpopulation in which the QTL formechanical stalk strength were detected. The lines were sorted byphenotype and assessed at the following marker loci: PHM2130, PHM15089,PHM12706, PHM5421, PHM201, PHM12521, PHM10840, PHM3468, PHM16736,PHM14053, PHM405, and PHM12025. Table 5 shows the individualpolymorphisms associated with increased mechanical stalk strength(“favorable”; select for) or decreased mechanical stalk strength(“unfavorable”; select against).

TABLE 5 In reference Identifier SNP Position sequenceChromosome 1 - QTL1A Select against: PHM2130.24 T  75 SEQ ID NO: 202PHM2130.29 A 170 SEQ ID NO: 202 PHM2130.30 C 179 SEQ ID NO: 202PHM2130.33 G 358 SEQ ID NO: 202 Chromosome 1 - QTL1B Select against:PHM15089.13 G 284 SEQ ID NO: 205 Chromosome 1 - QTL1C Select against:PHM12706.14 C 322 SEQ ID NO: 210 Chromosome 5 Select for: PHM5421.5 G314 SEQ ID NO: 12 PHM3468.1 G  93 SEQ ID NO: 15 PHM3468.4 T 101SEQ ID NO: 15 PHM3468.18 G 245 SEQ ID NO: 15 PHM12521.12 T 101SEQ ID NO: 14 PHM12521.19 A 186 SEQ ID NO: 14 PHM10840.105 C  37SEQ ID NO: 16 PHM10840.118 A 240 SEQ ID NO: 16 PHM10840.130 C 315SEQ ID NO: 16 Select against: PHM201.10 C 132 SEQ ID NO: 10 PHM201.18 A230 SEQ ID NO: 10 Chromosome 9 Select for: PHM16736.6 C 225SEQ ID NO: 199 PHM16736.8 T 326 SEQ ID NO: 199 PHM16736.14 A 422SEQ ID NO: 199 PHM14053.7 C 193 SEQ ID NO: 198 PHM14053.8 C 341SEQ ID NO: 198 PHM14053.14 C 386 SEQ ID NO: 198 PHM405.35 T 374SEQ ID NO: 212 PHM12025.26 C 216 SEQ ID NO: 213

Example 2 QTL5 Detection: Composite Interval Mapping

A composite interval mapping approach that combines interval mappingwith linear regression was undertaken to identify maize chromosomalintervals and markers associated with mechanical stalk strength. In aninterval mapping approach (Lander and Botstein, Genetics 121:185-199(1989)), each of many positions along the genetic map (say at 1 cMintervals) is tested for the likelihood that a QTL is located at thatposition. The genotype/phenotype data are used to calculate for eachtest position a LOD score (log of likelihood ratio). When the LOD scoreexceeds a threshold value (herein the threshold value is 2.5), there issignificant evidence for the location of a QTL at that position on thegenetic map (which will fall between two particular marker loci).

A high resolution genetic mapping population, the intermated B73×Mo17(IBM) population, was created by Lee, M et al., Plant Mol Biol48:453-461 (2002), and is a widely used resource for maize mapping. TheB73 inbred represents the Iowa Stiff Stalk Synthetic (BSSS) heteroticgroup, whereas Mo17 represents a non-BSSS heterotic group. Thepopulation was developed by intermating the F₂ for four generations andthen deriving recombinant inbred lines.

Recombinant inbred lines from the IBM syn4 population were obtained forthis study, and two sets of data were collected for each of the 272individuals obtained. The first set of data was collected using aportable Instron™ machine, near flowering. One growing season with twofield replications was used. Measurements were taken at nodes 3 and 4below the ear and then averaged over replications. The second set ofdata was obtained using the device and method described in patentapplication US2007/0125155 (published Jun. 6, 2007). Data were collectednear flowering, and the unit of measure was the number of plants thatexhibited snapping at a node. Both sets of data were representative ofmechanical stalk strength at the vegetative stage, or resistance tobrittle snap.

The 272 individuals of the IBM Syn4 generation were genotyped using 324markers, and the mean scores (averaged across replications; 2 replicatesfor the Instron™ data, 3 replicates for phenotypic analysis usingartificial wind stimulus) were input into Windows QTL Cartographer.Windows QTL Cartographer (the most up-to-date version of this softwarewas used at the date of QTL mapping) was used to perform the compositeinterval mapping. LOD scores (logarithm of the odds ratio) wereestimated across the genome according to standard QTL mappingprocedures.

For both sets of data, the composite interval mapping analysis showedone major significant QTL on chromosome 5 (FIG. 7 and FIG. 8), asdefined by a significance LOD score threshold of 2.5. The linkage mapused for composite interval mapping was a modified IBM2 map for whichthe genetic distances correspond to a single meiosis recombinationfraction (this map was generated internally). The modified IBM2 mapcomprised the following PHM marker loci located within the chromosome 5region of interest (with genetic map positions in parentheses): PHM5266(70.56 cM), PHM4115 (73.37 cM), PHM12521 (74.86 cM), PHM10840 (76.53cM), PHM4103 (77.29 cM), PHM9518 (78.89 cM), PHM4736 (81.94 cM), PHM6441(82.51 cM), and PHM430 (86.77 cM). For data set 1 (portable Instron™data; near flowering; referred to herein as CIM1 for “composite intervalmapping data set 1”), the location of the QTL encompassed markersPHM12521, PHM10840, PHM4103, and PHM9518. For data set 2 (wind machine;near flowering; referred to herein as CIM2 for “composite intervalmapping data set 2”), the location of the QTL encompassed markersPHM5266, PHM4115, PHM12521, PHM10840, PHM4103, PHM9518, PHM4736,PHM6441, and PHM430.

Example 3 Summary of QTL5 Studies

The results of the chromosome 5 analyses are summarized in Table 6 (themap position of each of the PHM markers on the internally derivedgenetic map is provided as a reference). All three studies identify thelocation of a QTL for mechanical stalk strength in the same region ofchromosome 5.

TABLE 6 Summary of QTL5 Studies Relative PHB Marker map position Locus(cM) Association CIM1 CIM2 PHM5266 70.56 X PHM4115 73.37 ** X PHM1252174.86 ** X X PHM10840 76.53 ** X X PHM4103 77.29 * X X PHM9518 78.89 * XX PHM4736 81.94 X PHM6441 82.51 X PHM430 86.77 X ** = p < 0.001; * = p <0.01

A set of common markers can be used to establish a framework foridentifying markers in the QTL interval (Table 7). In this table, themarkers shaded in gray are public markers, while the unshaded markersare provided herein. All of the markers listed in this table are in aconsistent position relative to one another on the PHB internallyderived map, the IBM2 neighbors genetic map, and the current physicalmap (FIGS. 1A-I). In FIGS. 1A-I, PHM12521 and PHM3468 are not shownbecause the markers are currently on an “unknown” contig that is notassembled to the larger chromosome 5 contig.

TABLE 7 Molecular marker positions on the PHB map and the IBM2 Neighborsmap

Markers shaded in gray are public markers.

Example 4 QTL Validation

Biparental crosses are typically created to validate QTLs. For example,near isogenic lines containing the individual QTL or combinations of QTLcan be generated from a biparental cross of two lines (each with adistinct genotype at a QTL of interest), and the resulting plants can beevaluated for mechanical stalk strength. Phenotypes can be measuredusing an Instron™ machine to perform the three-point bend test duringlate season, a portable Instron™ to perform the three-point bend test ator near flowering, the device and method described in patent applicationUS2007/0125155 (published Jun. 6, 2007), or any other method in the artthat can be used to evaluate mechanical stalk strength. The lines arethen sorted based on their phenotypic values.

Production markers can be developed in the QTL region. These markerswill distinguish the parents from one another, preferably using a highthroughput assay, and are used to genotype the segregating progenyplants. Production markers could be developed, for example, fromindividual SNPs that distinguished lines with increased mechanical stalkstrength from lines with decreased mechanical stalk strength in theassociation study described in Examples 1A-E. For instance, PHM5421-5-V,PHM12521-19-U, and PHM16736-8-V were developed from SNPs PHM5421.5,PHM12521.19, and PHM16736.8, respectively.

The PHM markers could also be used to genotype the progeny via thesequencing of PCR products. The primers for each of the PHM marker lociand the corresponding reference sequence ID numbers are shown inTable 1. For PHM marker analysis, nested PCR reactions are performed,using the external and internal primers for each PHM marker. In thefirst PCR reaction, 0.90 μl of 10×PCR buffer, 0.18 μl of 10 mM dNTP mix,0.27 μl of 50 mM MgCl₂, 1.50 μl of 2.5 μM external forward primer, 1.50μl of 2.5 μM external reverse primer, 0.04 μl of Platinum Taq, 1.61 μlof ddH2O, and 3 μl of 1.5 ng/μl DNA are used. The thermocyclingconditions constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55°C. for 30 seconds, and 72° C. for 2 minutes, repeated for 24 cycles; 72°C. for 10 minutes; and a hold at 4° C. The DNA produced from the firstround of PCR is then diluted 1:9 with TE for use in the second round ofPCR. The reaction mix for the second round of PCR is the same except theinternal sets of primers are used, and the DNA is the diluted DNA fromthe first round of PCR. The thermocycling conditions for the secondround of PCR constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55°C. for 30 seconds, and 72° C. for 2 minutes, repeated for 28 cycles; 72°C. for 10 minutes; and a hold at 4° C. The PCR products are thensequenced directly.

Each marker locus is examined, and standard statistical analysis, suchas the student t-test or Kolmogorov-Smirnov test, can be used todetermine if the trait distributions for each allele are significantlydifferent from one another. If significant, the effect of this QTL onmechanical stalk strength would be validated.

Example 5 Validation of QTLS, QTL9, and QTL1A

A biparental population, PHO7H×PH7CM, was developed for the purpose ofvalidating QTLS, QTL9, and QTL1A. PHO7H had a score of 19.569 in theassociation study and carries the favorable alleles at the QTL5 markerloci, PHM5421, PHM201, PHM12521, PHM10840, and PHM3468; at the QTL9marker loci, PHM16736, PHM14053, PHM405, and PHM12025; and at the QTL1Amarker locus PHM2130. PH7CM had a score of 7.596 and did not carry thefavorable alleles at QTL5, QTL9, and QTL1A. F₂ individuals weregenotyped, and siblings carrying only the favorable alleles at eitherQTL5, QTL9, or QTL1A were selected. The selected siblings were selfedtwice before phenotyping F₄ individuals with an Instron™ machine afterflowering. Plants carrying favorable alleles at QTL1A showed a greaterpositive effect on load at yield, dry weight, and diameter; however,favorable alleles at QTL5 increase stalk strength without a significantchange in dry weight or diameter. No difference in stalk strength, dryweight, and diameter were seen amongst the siblings for the chromosome 9segment. Data for stalk strength as measured by the Instron™ is shown inTable 8.

TABLE 8 median median (favorable (unfavorable p-value allele) allele)QTL1A 3.81E−11 19.48 16.96 QTL5 6.49E−04 19.58 17.83 QTL9 1.66E−01 19.6820.39

The apparent lack of association between QTL9 and mechanical stalkstrength was resolved in another biparental population that wasdeveloped from a cross between PH891 and PH7CM. PH891 had a score of14.863 in the association study and also carries the favorable allelesat QTL5, QTL9, and QTL1A. NILs (near isogenic lines) carrying only asubsegment of the chromosome 9 region were created. A subsegment of thechromosome 9 QTL from PH7CM, in the region between 174.3 and 179 cM, hadan overall positive effect on mechanical stalk strength, while asub-segment of the chromosome 9 QTL from PH7CM in the region between 179and 181 had an overall negative effect on mechanical stalk strength.This explains why there was no net gain in observed stalk strengthamongst the F₄ siblings (described in the previous paragraph) thatcarried both segments together.

Example 6 Markers for Use in MAS of Plants with Increased MechanicalStalk Strength

A set of production SNP markers specific for each chromosomal region hasbeen developed (Table 9), and when used together, the markers can beused to identify the proper haplotype for the regions associated withmechanical stalk strength. The markers were developed by comparing thegenotypes and phenotypes at a number of PHM marker loci in the intervalsdescribed herein (QTL1A, QTL1B, QTL5, and QTL9) for a parental panel ofinbreds from NSS germplasm plus a few diverse lines. Markers werecreated using Invader Plus™ chemistry.

TABLE 9 Production Markers for Use In MAS PHB map Select Select MarkerQTL position (cM) For: Against: PHM18693-9-U 1A 106.7 T PHM10786-11-U 1A107.8 G PHM10786-5-U 1A 107.8 C PHM10786-6-U 1A 107.8 T PHM8057-801-U 1A110.2 G PHM4044-11-U 1B 132.4 T PHM14080-16-V 1B 133.2 A PHM15089-10-U1B 133.7 C PHM9364-6-U 1B 135.5 G PHM201-16-U 5 71.6 C PHM201-17-U 571.6 C or G PHM4861-20-U 5 71.6 T or G PHM4861-21-U 5 71.6 A PHM5421-5-V5 72.2 G PHM4115-35-U 5 73.4 G or T PHM12521-18-U 5 74.9 T PHM12521-19-U5 74.9 A PHM12521-29-U 5 74.9 G C00386-397-U 5 C PHM13418-18 9 174.37 CPHM13418-10 9 174.37 C PHM113-7 9 180.76 T PHM10337-11-U 9 181.2 T^(i)PHM16736-8-V 9 181.2 A PHM12025-48 9 179.78 C PHM11186-16-V 9 181.5 T

What is claimed: 1-8. (canceled)
 9. A method of identifying andselecting a maize plant that displays increased mechanical stalkstrength, the method comprising: a. detecting in the maize plant atleast one allele of a marker locus wherein (i) the marker locus islocated within a chromosomal interval comprising and flanked by PHM654and PHM6727, and (ii) the at least one allele is associated with ahaplotype comprising: (a) a “G” at PHM5421.5; (b) a “G” at PHM3468.1;(c) a “T” at PHM3468.4; and (d) a “G” at PHM3468.18; b. selecting amaize plant if said at least one allele is detected. 10-12. (canceled)13. The method of claim 9, wherein the marker locus is located within achromosomal interval comprising and flanked by PHM201 and PHM3323. 14.The method of claim 9, wherein the marker locus is located within achromosomal interval comprising and flanked by PHM201 and PHM3468. 15.The method of claim 9, wherein said at least one allele is: a. a “C” atPHM201-16-U; b. a “C” or a “G” at PHM201-17-U; c. a “T” or a “G” atPHM4861-20-U; d. an “A” at PHM4861-21-U; e. a “G” at PHM5421-5-V; f. a“G” or a “T” at PHM4115-35-U; g. a “T” at PHM12521-18-U; h. an “A” atPHM12521-19-U; i. a “G” at PHM12521-29-U; j. a “C” at C00386-397-U; k. a“T” at PHM12521.12; l. an “A” at PHM12521.19; m. a “C” at PHM10840.105;n. an “A” at PHM10840.118; or o. a “C” at PHM10840.30.
 16. A method ofidentifying and counter-selecting a maize plant that displays decreasedmechanical stalk strength, the method comprising: a. detecting in themaize plant at least one allele of a marker locus wherein: (i) themarker locus is located within a chromosomal interval comprising andflanked by PHM654 and PHM6727, and (ii) the at least one allele isassociated with a haplotype comprising: (a) a “C” at PHM201.10; and (b)an “A” at PHM201.18; and b. counter-selecting the maize plant if said atleast one allele is detected.
 17. The method of claim 16, wherein themarker locus is located within a chromosomal interval comprising andflanked by PHM201 and PHM3323.
 18. The method of claim 16, wherein themarker locus is located within a chromosomal interval comprising andflanked by PHM201 and PHM3468.
 19. A method of identifying and selectinga maize plant that displays increased mechanical stalk strength, themethod comprising: a. detecting in the maize plant at least one alleleof a marker locus wherein: (i) the marker locus is located within achromosomal interval comprising and flanked by PHM4578 and PHM11186, and(ii) the at least one allele is associated with a haplotype comprising:(a) a “C” at PHM16736.6; (b) a “T” at PHM16736.8; (c) an “A” atPHM16736.14; (d) a “C” at PHM14053.7; (e) a “C” at PHM14053.8; (f) a “C”at PHM14053.14; (g) a “T” at PHM405.35; and (h) a “C” at PHM12025.26; b.selecting a maize plant as having increased mechanical stalk strength ifsaid at least one allele is detected.
 20. The method of claim 19,wherein said at least one allele is: a. a “T” at PHM113-7; b. a “T” atPHM10337-11-U; c. an “A” at PHM16736-8-V; d. a “C” at PHM12025-48; or e.a “T” at PHM11186-16-V.
 21. The method of claim 19, wherein (i) themarker locus is located within a chromosomal interval comprising andflanked by PHM14053 and PHM16736, and (ii) the at least one allele isassociated with a haplotype comprising: (a) a “C” at PHM16736.6; (b) a“T” at PHM16736.8; (c) an “A” at PHM16736.14; (d) a “C” at PHM14053.7;(e) a “C” at PHM14053.8; (f) a “C” at PHM14053.14; and (g) a “T” atPHM405.35.
 22. The method of claim 21, wherein said at least one alleleis: a. a “T” at PHM113-7; b. a “T” at PHM10337-11-U; or c. an “A” atPHM16736-8-V.
 23. A method of identifying and counter-selecting a maizeplant that displays decreased mechanical stalk strength, the methodcomprising: a. detecting in the maize plant at least one allele of amarker locus wherein: (i) the marker locus is located within achromosomal interval comprising and flanked by PHM7844 and PHM8029, and(ii) the at least one allele is associated with a haplotype comprising:(a) a “T” at PHM2130.24; (b) an “A” at PHM2130.29; (c) a “C” atPHM2130.30; and (d) a “G” at PHM2130.33; and b. counter-selecting themaize plant if said at least one allele is detected.
 24. The method ofclaim 23, wherein said marker locus lies within a chromosomal intervalcomprising and flanked by PHM7844 and PHM574.
 25. A method ofidentifying and selecting a maize plant or germplasm with increasedmechanical stalk strength comprising: a. obtaining DNA accessible foranalysis; b. detecting the presence of at least one marker allele thatis linked to and associated with a haplotype comprising: i) a “T” atPHM18693-9-U, ii) a “G” at PHM10786-11-U, iii) a “C” at PHM10786-5-U,iv) a “T” at PHM10786-6-U, and v) a “G” at PHM8057-801-U; and c.selecting said maize plant or germplasm that has the at least one markerallele.
 26. A method of identifying and counter-selecting a maize plantor germplasm with decreased mechanical stalk strength comprising: a.obtaining DNA accessible for analysis; b. detecting the presence of atleast one marker allele that is linked to and associated with ahaplotype comprising: i) a “T” at PHM4044-11-U, ii) an “A” atPHM14080-16-V, iii) a “C” at PHM15089-10-U, and iv) a “G” atPHM9364-6-U; and c. counter-selecting said maize plant or germplasm thathas the at least one marker allele.
 27. The method of claim 26, whereinthe at least one marker allele is linked to the haplotype by 20 cM. 28.The method of claim 26, wherein the at least one marker allele is linkedto the haplotype by 2 cM.